An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping

Area of Science:

• Neuroscience
• Optogenetics
• Molecular Biology

Background:

• Optogenetic tools like channelrhodopsin-2 (ChR2) are crucial for neural circuit manipulation and mapping.
• Current ChR2 variants lack selective transport to long-range axonal projections, leading to imprecise presynaptic activation and interpretation challenges due to spurious activation of en passant fibers.

Purpose of the Study:

• To engineer a ChR2 variant specifically localized to presynaptic axon terminals for precise activation.
• To improve the accuracy of neural circuit mapping and functional effect interpretation in optogenetic experiments.

Main Methods:

• Engineering of a ChR2 variant by fusing the metabotropic glutamate receptor 2 (mGluR2) C-terminal domain with a proteolytic motif and axon-targeting signal (mGluR2-PA tag).
• Localization of the mGluR2-PA-tagged ChR2-YFP at axon terminals.
• In vivo circuit connectivity mapping using the Spike Collision Test.

Main Results:

• The mGluR2-PA tag successfully localized ChR2-YFP to axon terminals without disrupting normal neural transmission.
• mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas, enabling photostimulation at lower intensities.
• The Spike Collision Test demonstrated that mGluR2-PA-tagged ChR2 is effective for identifying axonal projections with a significant reduction in polysynaptic noise.

Conclusions:

• The mGluR2-PA tag facilitates trafficking of ChR2 to axon terminals, enabling precise presynaptic activation.
• This engineered ChR2 variant enhances the accuracy of neural circuit mapping by minimizing spurious activation and polysynaptic noise.
• The developed tool offers significant potential for advancing optogenetic experiments and understanding neural circuit function.