Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination

Taro Yamanashi,Akihiro Yamanaka,Keiichi Kojima,Misayo Maki,Takashi Tsukamoto,Atsushi Shibukawa,Shin Takagi,Srikanta Chowdhury,Yuki Sudo

doi:10.1038/s41598-019-44308-x

Taro Yamanashi, Akihiro Yamanaka + Show 7 more

Open Access

https://doi.org/10.1038/s41598-019-44308-x

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Abstract

Ion pumps and channels are responsible for a wide variety of biological functions. Ion pumps transport only one ion during each stimulus-dependent reaction cycle, whereas ion channels conduct a large number of ions during each cycle. Ion pumping rhodopsins such as archaerhodopsin-3 (Arch) are often utilized as light-dependent neural silencers in animals, but they require a high-density light illumination of around 1 mW/mm2. Recently, anion channelrhodopsins -1 and -2 (GtACR1 and GtACR2) were discovered as light-gated anion channels from the cryptophyte algae Guillardia theta. GtACRs are therefore expected to silence neural activity much more efficiently than Arch. In this study, we successfully expressed GtACRs in neurons of the nematode Caenorhabditis elegans (C. elegans) and quantitatively evaluated how potently GtACRs can silence neurons in freely moving C. elegans. The results showed that the light intensity required for GtACRs to cause locomotion paralysis was around 1 µW/mm2, which is three orders of magnitude smaller than the light intensity required for Arch. As attractive features, GtACRs are less harmfulness to worms and allow stable neural silencing effects under long-term illumination. Our findings thus demonstrate that GtACRs possess a hypersensitive neural silencing activity in C. elegans and are promising tools for long-term neural silencing.

Highlights

In environments on the earth, there are a variety of ions and organisms have ion transporters on their cell membranes that transport ions such as H+, Na+, K+ and Cl−
An H+ pump in mitochondria produces an H+ gradient across the cell membrane that is necessary for the synthesis of adenosine triphosphate (ATP)[2], while the influx of Na+ into a cell by a Na+ channel triggers the action potential required for cell-to-cell communication, especially neurotransmission[3]
The results demonstrate that the light intensity required for GtACRs to sufficiently cause locomotion paralysis was three orders of magnitude smaller than that required for Arch

Summary

Results and Discussion

Functional expression of GtACR1 and GtACR2 in C. elegans neurons. To confirm the protein expression levels of GtACR1 and GtACR2 in C. elegans, the gene for green fluorescent protein (eGFP) was fused to the C-termini of GtACR1 and GtACR2 (Fig. S1). Transgenic worms expressing GtACR1 or GtACR2 opsins without all-trans retinal (ATR), an essential cofactor for rhodopsin, showed no locomotion paralysis regardless of the presence of illumination (Supplementary Video 2) These results suggest that, in the presence of ATR, GtACR1 and GtACR2 are functionally expressed in C. elegans neurons and effectively silence motor neurons governing contraction and relaxation of body wall muscles as previously demonstrated for Arch[14]. The normalized slope of body length change per sec for GtACR2 was comparable to that of Arch, suggesting that GtACR2 efficiently induces neural silencing in freely moving C. elegans. GtACR1 and GtACR2 efficiently induced locomotion paralysis of the worms at a roughly 3 orders of magnitude lower light intensity compared with Arch These results demonstrate that GtACRs have ultra-efficient neural silencing activities in C. elegans. We believe that the advantages of GtACRs would be of high interests for neuroscientists

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