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


Autoři: Masaaki Kuwajima aff001;  Olga I. Ostrovskaya aff001;  Guan Cao aff001;  Seth A. Weisberg aff002;  Kristen M. Harris aff001;  Boris V. Zemelman aff001
Působiště autorů: Center for Learning and Memory, The University of Texas at Austin, Austin, Texas, United States of America aff001;  Department of Neuroscience, The University of Texas at Austin, Austin, Texas, United States of America aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226797

Souhrn

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.

Klíčová slova:

Axons – Electron microscopy – Functional electrical stimulation – Light pulses – Neurons – Synapses – Immunogold labeling – Glutaraldehyde fixation


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