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Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System.

Jun-Young Oh1, Jeong-Ho Han2, Hyoeun Lee2

  • 1Multi-institutional Collaborative Research Center for Cortical Processing, Korea Brain Research Institute (KBRI), Daegu 41062, Korea.

Experimental Neurobiology
|July 7, 2020
PubMed
Summary

Researchers developed a new neural circuit tracing system, cFork, to simultaneously label postsynaptic neurons. This system enables detailed mapping of multisynaptic inputs, advancing our understanding of brain function and connectivity.

Keywords:
Adeno-associated virus -1Adeno-associated virus -5Anterograde tracingBrain mappingEGFPmScarlet

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Genetics

Background:

  • Understanding neural circuit connectivity is crucial for decoding brain functions.
  • Trans-synaptic viral vectors aid in mapping neural connections but cannot label multisynaptic inputs simultaneously.
  • Current methods lack the ability to concurrently trace multiple presynaptic inputs to individual postsynaptic neurons.

Purpose of the Study:

  • To develop a novel neural circuit tracing system capable of simultaneously labeling postsynaptic neurons based on their presynaptic input connectivity.
  • To overcome the limitations of existing trans-synaptic viral vectors in mapping complex neural circuits.
  • To provide a tool for detailed analysis of multisynaptic connectivity in the brain.

Main Methods:

  • Developed the "cFork" system using adeno-associated virus serotype 1 (AAV1) vectors carrying Cre or flippase recombinase (FlpO) delivered to presynaptic areas.
  • Utilized AAV5 vectors with a dual gene expression cassette in postsynaptic neurons for differential labeling.
  • Validated the system in vitro and in vivo, including applications in the somatosensory barrel field cortex (S1BF) and striatum.

Main Results:

  • Successfully achieved simultaneous labeling of postsynaptic neurons with two distinct fluorescent proteins (EGFP and mScarlet) based on presynaptic input.
  • Demonstrated anterograde trans-synaptic transfer of Cre or FlpO constructs via AAV1 vectors.
  • Showcased the system's efficacy in tracing complex multisynaptic inputs in specific brain regions.

Conclusions:

  • The cFork system offers a novel method for simultaneous, input-defined labeling of postsynaptic neurons.
  • This tool facilitates the elucidation of complex multisynaptic connectivity patterns.
  • cFork has the potential to significantly advance research into the neural basis of diverse brain functions.