Nearest neighbor analysis of dopamine D1 receptors and Na⁺‐K⁺‐ATPases in dendritic spines dissected by STED microscopy

Abstract

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na(+),K(+)-ATPase. The analysis gave new information on how dense the D1 receptor and Na(+),K(+)-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na(+),K(+)-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes.

Lens-based fluorescence nanoscopy

“…(2) Nanoscopic co-localization studies of various different proteins have been enabled by a carefully optimized choice of two conventional Stokes-shifted fluorophores, whose spectra differ by only about 60 nm, the use of two pairs of excitation/ STED lasers timed in a pulsed interleaved excitation scheme, and with an elimination of the detection cross-talk by applying linear unmixing algorithms (Fig. 3c, d) (Blom et al 2012;Dean et al 2012;Neumann et al 2010;Opazo et al 2010;Reisinger et al 2011;Osseforth et al 2013). In this scheme, the number of distinguishable labels could be increased to four by separating the emission based on emission wavelength and lifetime (3) Recent two-color STED imaging has been realized using two excitation and a single STED laser only, e.g.…”

“…Scale bar: 500 nm. (c) Multi-color STED nanoscopy determining the co-localization of different molecules with sub-diffraction resolution, as exemplified for the D1 dopamine receptor and Na1,K1-ATPase in cultured striatal neurons (lower image: confocal recording, adapted from (Blom et al 2012)). Scale bars: 1 μm (in enlarged image: 200 nm).…”

Recent applications of superresolution microscopy in neurobiology

“…One-color and later two-color STED microscopy revealed the clustered distribution of another synaptic protein, the a3 isoform of the Na + ,K + -ATPase, which is responsible for the active transport of Na + and K + ions across the plasma membrane and is purported to play a role in excitatory synapses [14]. Subsequently, these authors studied the distribution of Na + ,K + -ATPase in relation to the dopamine D1 receptor in dendritic spines and applied nearest neighbor analysis to support the finding of both a co-localized and a non co-localized confinement of the receptor and the ionic pump macromolecules in dendritic spines of cultures striatal neurons [15]. A weak overlap albeit clustered distribution was found for the low-abundance phosphoprotein DARPP-32 with the dopamine D1 receptor in spines of striatal neurons [16].…”

Fluorescence Nanoscopy of Platelets Resolves Platelet‐State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications

“…The home-built STED microscope has been described in detail before [21] , but with a few minor differences [22] . In brief, two excitation beams ( …”