Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation

Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-ph...

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Bibliographic Details
Published in:PloS one 2020-01, Vol.15 (1), p.e0226797-e0226797
Main Authors: Kuwajima, Masaaki, Ostrovskaya, Olga I, Cao, Guan, Weisberg, Seth A, Harris, Kristen M, Zemelman, Boris V
Format: Article
Language:English
Subjects:
Animals
Axons
Axons - metabolism
Axons - physiology
Axons - radiation effects
Axons - ultrastructure
Back propagation
Biology and Life Sciences
Electron microscopy
Embedding
Experiments
Hippocampus
Laboratory animals
Light
Long-term potentiation
Long-Term Potentiation - radiation effects
Medicine and Health Sciences
Memory
Methods
Microscopy
Neurosciences
Optogenetics
Organelles
Organic chemistry
Physical Sciences
Potentiation
Preservation
Proteins
Rats
Research and Analysis Methods
Synapses
Synapses - metabolism
Synapses - radiation effects
Ultrastructure
Viruses
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Summary:Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-phase long-term potentiation (L-LTP) in area CA1. L-LTP was shown to be specific to the labeled axons by severing CA3 inputs, which prevented back-propagating recruitment of unlabeled axons. Membrane-associated mAPEX2 tolerated microwave-enhanced chemical fixation and drove tyramide signal amplification to deposit Alexa Fluor dyes in the light-activated axons. Subsequent post-embedding immunogold labeling resulted in outstanding ultrastructure and clear distinctions between labeled (activated), and unlabeled axons without obscuring subcellular organelles. The gold-labeled axons in potentiated slices were reconstructed through serial section electron microscopy; presynaptic vesicles and other constituents could be quantified unambiguously. The genetic specification, reliable physiology, and compatibility with established methods for ultrastructural preservation make this an ideal approach to link synapse ultrastructure and function in intact circuits.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0226797