Elsevier

Neuroscience Research

Volume 75, Issue 1, January 2013, Pages 69-75
Neuroscience Research

Technical note
Targeted expression of a chimeric channelrhodopsin in zebrafish under regulation of Gal4-UAS system

https://doi.org/10.1016/j.neures.2012.08.010Get rights and content

Abstract

Channelrhodopsin (ChR)-wide receiver (ChRWR), one of the chimeric molecule of ChR1 and ChR2, has several advantages over ChR2 such as improved expression in the plasma membrane and enhanced photocurrent with small desensitization. Here we generated transgenic zebrafish (Danio rerio) expressing ChRWR as a conjugate of EGFP under the regulation of UAS promoter (UAS:ChRWR-EGFP). When crossed with a Gal4 line, SAGFF36B, ChRWR-EGFP was selectively expressed in primary mechanosensory Rohon–Beard (RB) neurons. The direct photoactivation of RB neurons was sufficient to trigger the escape behavior. The UAS:ChRWR-EGFP line could facilitate a variety of investigations of neural networks and behaviors of zebrafish in vivo.

Highlights

ChRWR is optimized for stimulating a neuron with low intensity of light. ► We generated UAS:ChRWR-EGFP line of zebrafish expressing ChRWR under UAS promoter. ► When crossed with an SAGFF36B line, ChRWR was expressed in Rohon–Beard neurons. ► The escape behavior was elicited by photoactivation of Rohon–Beard neurons. ► The UAS:ChRWR-EGFP line is an useful tool to study neuronal network of zebrafish.

Introduction

The zebrafish (Danio rerio) nervous system is an ideal vertebrate model to investigate the relationship between neural activity and behavior. Each neuron is identifiable in the living fish larva because of its transparency. The fundamental behaviors of vertebrates are also conserved in the zebrafish (Portugues and Engert, 2009, McLean and Fetcho, 2011). The Gal4-UAS system, which was first designed as a two-component gene expression in Drosophila (Brand and Perrimon, 1993), enables one to target the transgene expression to specific types of neurons in the zebrafish brain (Scheer and Campos-Ortega, 1999, Jeong et al., 2007, Scott, 2009). Recently, various Gal4-driver lines with specific expression patterns have been established using enhancer trap methods (Asakawa and Kawakami, 2008, Kawakami et al., 2010).

To understand what neuronal activity is sufficient for evoking behavior, direct stimulation of specific neurons in live animal models should be useful. Optical stimulation methods have advantages over conventional electrical stimulation methods because of their fine spatiotemporal resolution, relative harmlessness and convenience (Callaway and Yuste, 2002, Miesenböck, 2004). Recently photostimulation using channelrhodopsin-2 (ChR2) has become a powerful tool for the investigation of neural networks both in vivo and in vitro (Boyden et al., 2005, Li et al., 2005, Ishizuka et al., 2006, Bi et al., 2006, Douglass et al., 2008). Expressing ChR2 selectively in a genetically identified group of neurons, a brief blue light irradiation depolarized them over the threshold to evoke an action potential without directly affecting the other neurons. Although ChR2 has been widely applied for the photostimulation of neurons, recent technological developments have made it possible to select ChR variants optimized to the particular experiment (Mattis et al., 2012).

Channelrhodopsin-wide receiver (ChRWR), one of chimeric channelrhodopsins (ChRs) which consists of 1–245 amino acids from channelrhodopsin-1 (ChR1) and 208–315 amino acids from ChR2, has several advantages over ChR2 such as improved expression in the plasma membrane and an enhanced photocurrent with small desensitization (Wang et al., 2009). In this study, we report on the establishment of a reporter zebrafish line, UAS:ChRWR-EGFP. We found the expression of ChRWR in specific types of neurons as expected from the driver Gal4 lines with the specific behavioral responses evoked by the photostimulation.

Section snippets

Cell culture and molecular biology

The ChRWR plasmids of a C-terminal fusion construct with Venus (Nagai et al., 2002) was made as previously described (Wang et al., 2009). Human embryonic kidney (HEK) 293 cells were grown in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum under a 5% CO2 atmosphere at 37 °C. The expression plasmids were transiently transfected in HEK293 cells using Effectene transfection reagent (Qiagen, Tokyo, Japan) according to the manufacturer's instructions.

Results

The photocurrent kinetics of ChRWR was investigated as a function of the irradiance using the HEK293 expression system. As shown in Fig. 1A, the blue light pulses (460–490 nm, 5 s) evoked photocurrents from ChR2 and ChRWR in HEK293 cells in a manner dependent on the irradiance. The green light pulses (545–580 nm, 5 s) evoked relatively large ChRWR photocurrents although almost negligibly activated ChR2 photocurrent (Fig. 1B). The photocurrent amplitude of ChRWR was generally larger than that of

Discussion

In this study we found that the HEK293 cells expressing ChRWR generated relatively larger peak and steady-state photocurrents than those expressing ChR2. The effectiveness of ChRWR was manifest with small irradiance. This is in part attributed to the efficient membrane expression of ChRWR (Wang et al., 2009). In addition, the desensitization of the ChRWR photocurrent was relatively small (about 20% of the peak). Therefore, ChRWR would be optimal for experiments stimulating a neuron repetitively

Acknowledgements

We are grateful to K. Abe, T. Honjoh, S. Hososhima, T. Igarashi, H. Katow, K. Kikuchi, T. Mishima, T. Okazaki, S. Watanabe and Y. Yokoyama for experimental assistance and to B. Bell for language assistance. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Mesoscopic Neurocircuitry” (No. 23115501) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Global COE Program (Basic & Translational Research Center for Global Brain

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