An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping

Shun Hamada,Tomohiko Yoshizawa,Akari Hagiwara,Masashi Nagase,Yoshikazu Isomura,Toshihisa Ohtsuka,Ayako M Watabe

doi:10.1038/s42003-021-01977-7

Abstract

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron’s long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments.

Highlights

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits
We chose two presynaptic proteins, CAST and RIM1, which organize the synaptic vesicle release at the active zone (AZ) (Fig. 1a)[29,30]. As their full length was too large to insert as localization tags, we attempted to use the putative N-terminal coiled-coil domain of CAST (139–306 aa; ref. 31) and the RIM1 PDZ domain (614–693 aa), which is essential for interacting with CAST31,32 at the presynaptic terminal
Judging from the band size, the expression of ChR2-YFP was preserved with the CAST, metabotropic glutamate receptor 2 (mGluR2), and myosin VI binding domain (MVIBD) tags, whereas, unexpectedly, it was attenuated with the RIM1 tag (Fig. 1b, Supplementary Fig. 1a)

Summary

Introduction

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. The PDZ domain-binding motif, ETQV, concentrated ChR2 in the postsynaptic density[16] While these modifications are beneficial for activating target neurons more locally and efficiently, activation of long-range axonal projections is not well developed. ChR2 with a neurexin 1α tag promoted presynaptic enrichment, these reports did not describe validation of functional improvement of the channels.[21,22] It is still worth developing novel ChR2 tools specific to long-range axons and axon terminals that will be capable of eliciting spikes and synaptic release. Subcellular enrichment of ChR2 to distal axons and axon terminals, and lowering expression in somata and dendrites would be strategically beneficial for triggering antidromic spikes and synaptic release in intricate neural circuits. Such axonoriented ChR2 variants have not been established so far

Methods

Results

Conclusion