Abstract
An engineered light-inducible chloride pump, Natronomonas pharaonis halorhodopsin 3 (eNpHR3) enables temporally and spatially precise inhibition of genetically defined cell populations in the intact nervous tissues. In this report, we show the generation of new mouse strains that express eNpHR3-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to optically inhibit neuronal activity. In these mouse strains, Cre/Flp recombination induced high levels of opsin expression. We confirmed their light-induced activities by brain slice whole-cell patch clamp experiments. eNpHR3-expressing neurons were optically hyperpolarized and silenced from firing action potentials. In prolonged silencing of action potentials, eNpHR3 was superior to eNpHR2, a former version of the engineered pump. Thus, these eNpHR3 mouse strains offer reliable genetic tools for light-induced inhibiting of neuronal activity in defined sets of neurons.
Highlights
Optogenetic technics for artificially regulating neuronal activities by light are widely employed for manipulating the nervous system[1,2,3]
The main structure of the targeting vector is composed of a ubiquitous CAG promoter, LoxP-flanked www.nature.com/scientificreports primary and Frt-flanked secondary stop cassettes, and eNpHR3-EYFP
A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence was integrated into the 3′UTR region to enhance transgene expression levels by mRNA stabilization[17]
Summary
Optogenetic technics for artificially regulating neuronal activities by light are widely employed for manipulating the nervous system[1,2,3]. Optogenetic strategies use light to artificially regulate cellular functions in targeted cells in vivo with high temporal and spatial precision[4,5,6,7]. The original NpHR has undergone molecular modifications to improve its surface membrane localization and reduce intracellular aggregation by attaching endoplasmic reticulum export and neurite trafficking motifs[11,12]. These second- and third-generation enhanced tools (eNpHR2 and eNpHR3) have been successfully applied in vivo and in nervous system in a number of studies[13,14,15,16,17,18]. We compared the eNpHR3-knock-in mice to the previously generated eNpHR2-knock-in mice[19] under the same experimental conditions, and confirmed the superior ability of the eNpHR3-knock-in/BAC transgenic mice in light-triggered silencing of neuronal activities
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