TY - JOUR
T1 - Targeted expression of step-function opsins in transgenic rats for optogenetic studies
AU - Igarashi, Hiroyuki
AU - Ikeda, Keiko
AU - Onimaru, Hiroshi
AU - Kaneko, Ryosuke
AU - Koizumi, Kyo
AU - Beppu, Kaoru
AU - Nishizawa, Kayo
AU - Takahashi, Yukari
AU - Kato, Fusao
AU - Matsui, Ko
AU - Kobayashi, Kazuto
AU - Yanagawa, Yuchio
AU - Muramatsu, Shin Ichi
AU - Ishizuka, Toru
AU - Yawo, Hiromu
N1 - Funding Information:
The authors thank N. Copeland for providing the recombineering reagents, K. Kawakami, N. Takahashi and A. Morita for technical assistance, and B. Bell for language assistance. The authors also thank N. Takino and M. Ito for vector production. The authors (FK and YT) thank Drs. Kubota and Kawaguchi for advice on GABA immunostaining and Dr. Watabe for application process of VGAT-Cre BAC rats. This work was financially supported by a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; the JSPS KAKENHI (15J05551 to H.I., 16K07003 to H.O., 26290002, 15H05872, 15H01415, and 17H05550 to Y.Y., 25290002 to T.I., 25115701, 25250001, 25670103, 15K15025, and 15H01413 to H.Y.); the subsidies for private universities (Showa University of Medicine and Jichi Medical University); the Programme for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) to H.Y.; CREST and JST (Y.Y., T.I. and H.Y); The Ministry of Education, Culture, Sports, Science and Technology-Supported Program for the Strategic Research Foundation at Private Universities (S1311009) to F.K.; a Grant-in-Aid for Scientific Research (B) to F.K. (25293136); Strategic Research Program for Brain Sciences to F.K (E).; Grant-in-Aids for Young Scientists (B) and for Scientific Research (C) to Y.T (15K19194 and 17K09042); and the Tohoku University Division for Interdisciplinary Advanced Research and Education (DIARE) to H.I.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.
AB - Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.
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U2 - 10.1038/s41598-018-23810-8
DO - 10.1038/s41598-018-23810-8
M3 - Article
C2 - 29615713
AN - SCOPUS:85044974103
SN - 2045-2322
VL - 8
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 5435
ER -