In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex

Abstract

The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex. Adult knock-in mice expressing eGFP fused to the endogenous PSD95 protein were imaged at time points from 1 min to 6 h. Superresolved large assemblies of PSD95 show different sub-structures; most large assemblies were ring-like, some horse-shoe or figure-8 shaped, and shapes were continuous or made up of nanoclusters. The sub-structure appeared stable during the shorter (minute) time points, but after 1 h, more than 50% of the large assemblies showed a change in sub-structure. Overall, these data showed a sub-morphology of large PSD95 assemblies which undergo changes within the 6 hours of observation in the anaesthetised mouse.

Cell‐type‐specific visualisation and biochemical isolation of endogenous synaptic proteins in mice

“…This has allowed us to survey and numerically quantify PSD95-eGFP puncta in terms of their number, size and intensity at the whole brain scale (Zhu et al, 2018). In addition, the genetic fluorescent labelling makes it possible to carry out live imaging in the intact animal, or acute brain slices, to study the molecular dynamics of PSD95 (Wegner, Mott, Grant, Steffens, & Willig, 2018).…”

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…Altogether SRM has led to remarkable improvement in the assessment of the intracellular localization and trafficking of nanoparticles compared to conventional fluorescence microscopy, being a valuable tool to dissect internalization pathways in cultured cells and, potentially, in vivo. 130,131 [H2] Material stability The enhanced resolution of SRM allows us to go beyond the bare visualization of nano-objects and their location, and probe several material properties in the cellular environment. This is particularly relevant for complex molecular architectures, designed to be responsive and adaptive in the biological environment.…”

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling

“…These previous in vivo STED imaging studies examined dendritic features based on (over)expression of fluorescent proteins (FPs) either as volume labeling or to mark the actin network. A notable exception is the most recently reported application of in vivo STED imaging to a synaptic protein endogenously tagged with EGFP ( 27 ). The use of green or yellow FPs in STED nanoscopy, however, brings about increased hazards of tissue photodamage due to the higher photon energies of the required excitation and STED light in the green–yellow range.…”