Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes

Nat Commun. 2019 Sep 26;10(1):4377. doi: 10.1038/s41467-019-12372-6.

Abstract

Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Cells, Cultured
  • Diffusion
  • Disks Large Homolog 4 Protein / metabolism
  • Mice
  • Microfilament Proteins
  • Microscopy, Fluorescence / methods*
  • Nerve Tissue Proteins / metabolism
  • Neuronal Plasticity
  • Neurons / cytology
  • Neurons / metabolism*
  • Nucleic Acid Probes / chemistry
  • Nucleic Acid Probes / metabolism*
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism*
  • Rats, Sprague-Dawley
  • Synapses / metabolism
  • Synaptic Vesicles / metabolism

Substances

  • Disks Large Homolog 4 Protein
  • Microfilament Proteins
  • Nerve Tissue Proteins
  • Nucleic Acid Probes
  • Oligonucleotides
  • Shank3 protein, mouse
  • locked nucleic acid