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Related Experiment Video

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Viral Tracing of Genetically Defined Neural Circuitry
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Visualization of Selective Neural Pathways by Double Viral Infection Method.

Akiya Watakabe1

  • 1Laboratory for Molecular Mechanism of Brain Development, RIKEN Center for Brain Science, Wako, Japan. akiya.watakabe@riken.jp.

Methods in Molecular Biology (Clifton, N.J.)
|November 22, 2025
PubMed
Summary

Viral vector technology allows detailed visualization of neural pathways. A novel double viral vector method enables precise labeling of neuronal connections for comprehensive pathway mapping.

Keywords:
Adeno-associated viral vectorAxon collateralCorticothalamicLentiviral vectorNeuRet vectorRetrograde gene transferTET-Off systemTracer

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Genetics

Background:

  • Classic neural tracing techniques have limitations in visualizing complex neural pathways.
  • Viral vector technology has advanced neural tracing capabilities.
  • Development of efficient retrograde vectors is crucial for successful neural pathway visualization.

Purpose of the Study:

  • To explain a strategy and protocol for visualizing selective neural pathways using a double viral vector infection method.
  • To detail the application of lentiviral and adeno-associated viral (AAV) vectors combined with a TET-Off/TET-On system.
  • To achieve Golgi-like visualization of neurons, from dendrites to axon terminals.

Main Methods:

  • Utilized a combination of lentiviral vectors for retrograde gene transfer and AAV vectors.
  • Split the TET-Off/TET-On system across the two viral vectors.
  • Co-injected the vectors at the target and origin sites of the neural pathway.

Main Results:

  • Successfully labeled specific neuronal populations connecting the two injection sites.
  • Achieved enhanced transgene expression via the TET system, resulting in Golgi-like visualization.
  • Demonstrated specific labeling of corticothalamic cells, including their collateral axons.

Conclusions:

  • The double viral vector infection method provides unprecedented visualization of neural pathways.
  • This technique enables comprehensive mapping of neuronal connections, including intricate axonal projections.
  • The method offers a powerful tool for studying neural circuitry with high resolution.