Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes.
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.
|Year of Publication
2019 09 26
|PubMed Central ID
R24 MH106075 / MH / NIMH NIH HHS / United States
U01 MH106011 / MH / NIMH NIH HHS / United States
RM1 HG008525 / HG / NHGRI NIH HHS / United States
T32 GM087237 / GM / NIGMS NIH HHS / United States
P30 ES002109 / ES / NIEHS NIH HHS / United States
R01 NS087950 / NS / NINDS NIH HHS / United States
R01 MH112694 / MH / NIMH NIH HHS / United States