Fluorescent in situ hybridization of synaptic proteins imaged with super‐resolution STED microscopy

Abstract

Super-resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super-resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation-emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super-resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells.

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

“…This destructive effect due to heat treatment was clearly shown by super-resolution scanning near-field optical microscopy (SNOM) [7]. In contrast to DNA–DNA FISH of chromatin in cell nuclei mRNA FISH using appropriate oligonucleotides can be optimally performed at nearly physiological temperatures [8] and applied to super-resolution microscopy [8,9] together with immunostaining. Although the combination of DNA-FISH and immunostaining resulted in apparently well-maintained chromatin morphology [4], it should be considered that the usually preferred conventional preparation techniques in super-resolution microscopy determine the practical limits of application [10].…”

Super‐resolution imaging for cell biologists

“…E: STED and confocal images of fluorescence in situ hybridization of synaptophysin mRNA reproduced with permission from Fig. 1 of . F: 3D‐SIM imaging of DAPI stained nuclear DNA reproduced with permission from Fig.…”

Advances in imaging ultrastructure yield new insights into presynaptic biology

“…LTP also appears to induce an elongation of the presynaptic membrane that is coordinated with increasing complexity of postsynaptic spines, changes that depend on the priming factor Munc13–1 (Zhao et al, 2012a ). In addition to trafficking new molecular complexes from the cell body, neurons may also accomplish molecular plasticity through local protein translation, and STED imaging is now being employed to address this question (Zhang et al, 2014 ). Although there is now significant evidence supporting translation of synaptic transcripts in the cell soma and dendrites, evidence in support of translation in the presynaptic compartment after the synapse primarily comes from studies employing the local application of protein synthesis inhibitors (Hsiao et al, 2014 ; reviewed in Akins et al, 2009 ).…”

“…Presynaptically translated transcripts remain difficult to identify, but include the neuropeptide sensorin in Aplysia, and β-catenin in dissociated rat hippocampal cultures (Liu et al, 2003 ; Lyles et al, 2006 ; Wang et al, 2009 ; Taylor et al, 2013 ). Although fluorescent in situ hybridization (FISH) detected messenger RNA (mRNA) of the synaptic proteins Synaptobrevin and Synaptotagmin only in the soma and occasional dendrite of cultured hippocampal neurons, applied to other synaptic proteins in diverse neuronal subtypes, the increased resolution gained from STED-FISH may reveal previously overlooked mRNA localization in presynaptic terminals (Zhang et al, 2014 ). Parallel ultrastructural studies employing cryopreservation and tomography may hold promise in addressing the failure to detect polyribosomes presynaptically.…”