Targeted expression of stepfunction opsins in transgenic rats for optogenetic studies

Abstract 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

A Novel Reporter Rat Strain That Conditionally Expresses the Bright Red Fluorescent Protein tdTomato

<div><p>Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic <i>Cre</i> mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both <i>in vivo</i> and <i>ex vivo</i>. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.</p></div

tdTomato expression in the brain of FLAME.

<p>(A) A sagittal section of the brain dissected from adult FLAME (n = 2). tdTomato was expressed ubiquitously in neurons throughout the brain, such as those in the olfactory bulb (B), the striatum (C), the hippocampus (D), the cerebral cortex (E), and the cerebellum (F).</p

Three-dimensional imaging of hippocampal CA1 after tissue clearing.

<p>(A) Three-dimensional reconstruction of the CA1 region of the hippocampus, which was treated with CUBIC reagent 1 for 2 days and imaged under two-photon microscopy. The laminar structure of pyramidal cells was obvious. Axons and dendrites were brightly labeled by tdTomato and easily traced throughout the tissue. (B) X–Y, X–Z, and Y–Z planar sections of the image shown in (A).</p

Specific expression of tdTomato in phox2b-Cre/floxed STOP tdTomato double transgenic rats.

<p>(A) Plain image (i) and tdTomato fluorescence image (ii) from whole embryos (E12.5): the oculomotor (III) and trochlear (IV) motor nuclei in the central nervous system (III and IV), and the three epibranchial sensory/distal ganglia; geniculate VII, petrosal IX, and nodose X ganglia (gVII, gIX, and gX, respectively). Note that the axons of spinal accessory nucleus (arrowhead) and neurons in the ventral neural tubes (arrow) are also fluorescent. (B, C) Brainstem of neonatal rat: tdTomato (i) and anti-Phox2b (ii). nTS, sensory neurons of the nucleus of the solitary tract; dmnX, dorsal motor nucleus of the vagus nerve; nA, nucleus ambiguus; AP, area postrema; VII, facial nucleus; RTN/pFRG, retrotrapezoid nucleus/parafacial respiratory group. (D) Co-localization of tdTomato and Phox2b: from left to right, tdTomato, anti-Phox2b, DAPI, and the merge.</p

Reliability of the reporter system.

<p>(A) Primary fibroblasts prepared from the tail tip of the floxed STOP tdTomato reporter transgenic rats; the expressions of AcGFP-NCre by lipofection (i), tdTomato fluorescence (ii), and merged images (iii) are shown. (Bi–ii) Primary fibroblasts cultured from Tg-positive rats treated with vehicle alone. (C) Comparison of the ratio of tdTomato/AcGFP double-positive cells in each group (Tg, <i>n</i> = 5; wild-type, <i>n</i> = 6). Ten fields of interest were unintentionally chosen from each well, and double-blinded counting was applied to detect the fluorescent double-positive cells. In total 219 double positive cells were detected in group (A). *, <i>p</i> < 0.01, one-way ANOVA with Scheffe’s multiple comparison test. (D) PCR assay of the genomic DNA each extracted from the tail and the brain of a wild-type control rat (left 2 lanes), a tdTomato reporter rat (middle 2 lanes) or a FLAME (right 2 lanes). The allele of BAC with STOP sequence was detectable as 497 bp band whereas that without STOP as 217 bp band.</p