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.

Figure 1

PSD95 shape and size determination in vivo. (A) Confocal and (B) STED microscopy image of PSD95-eGFP in L1 of the binocular mouse visual cortex (6 µm below the cover glass). The PSD95 morphology is very diverse: Some spots are very small and dark (1), some are elongated, stripe-like (2), and some are large protein assemblies (3a–c). (C) Magnification of large protein assemblies marked in (A), imaged with confocal microscopy. (D) Superresolution STED microscopy of the same assemblies as in (C) reveals a complex sub-structure which is not visible in the confocal image. 3a and 3b show a ring-like morphology whereas 3c shows three distinct clusters. (E) Absolute frequency histogram of PSD95 assembly size analysed from in vivo STED microscopy images, average size ± SD. 708 assemblies of n = 4 mice were analysed.

Figure 2

Morphology of large PSD95 assemblies changes within hours. (A–D) STED microscopy image of a selection of large protein assemblies recorded at different time intervals in the visual cortex of an anaesthetised mouse. The morphology is very diverse; many assemblies are smooth ring-like (A1, B1, B2 at t = 0, C1, D1, D2, D3), some are clustered (A2, B2 at 1.5 h, C2, D4); the cluster can be arranged in horse-shoe (A2), ring-like (C2 at 2 h, D4 at 6 h) or arbitrary (B2 at 1.5 h, C2 at 0 h, D4 at 0 h) shape. Shape changes were categorized as no changes (green), subtle changes (yellow) or strong morphological changes (red). All changes refer to t = 0. (A) Within minutes the morphology is not changing. (B) After 0.5 h assemblies can be rather stable (B1) or change strongly (B2). (C) Over 2 h assemblies can be stable (C1) or undergo morphological change (C2). (D) Assemblies can change and reverse to the original structure (D1), are stable after 3 h and changed after 6 h (D2), or undergo multiple changes (D3, D4). (E) Stacked histogram of relative frequencies of morphological changes of PSD95 assemblies; changes refer always to t = 0. Number of protein assemblies analysed: 18 (1 min), 18 (2 min), 13 (3 min), 43 (0.5 h), 43 (1 h), 35 (1.5–2 h), 19 (3–4 h), 10 (5–6 h) of n = 4 mice. All analysed protein assemblies can be found in the supplementary data. Scale bars 500 nm.

Figure 3

Mapping PSD95 assemblies over several hours in an anaesthetised mouse. (A,B,C) Maximum intensity projections of STED microscopy z-stacks at different areas in the visual cortex which were repetitively imaged at different time intervals of 1 min, 0.5 h and 2 h. (A’) RGB overlay of three binary images created from maximum intensity projections (MIP) at time point 0 (shown in A) (red), after 1 min (blue) and after 2 min (green). (B’) RGB overlay of binary images of MIPs recorded at t = 0 (created from B) (red), t = 0.5 h (blue), and t = 1 h (green). (C’) RGB overlay of binary images of MIPs recorded at t = 0 (created from C) (red), t = 2 h (blue) and t = 4 h (green). Overlapping colour areas are as indicated on each colour wheel. (A”–C”) Percent of pixels which do not change between two time points averaged over the whole binary image above. (A”) ~60% of the pixel of all assemblies in A’ at t = 0 overlap with the assemblies at t = 1 min (0–1 min) and correspondingly between t = 1 min and t = 2 min (1–2 min) and t = 0 min with t = 2 min (0–2 min). (B”) Only <40% of pixels of the assemblies in B’ show an overlap between 0 and 1 h, and ~20% after 4 h (C”). Scale bars 2 µm.