Transgene expression of the mhChR2::YFP fusion protein is directed to serotonergic neuronal populations by the mouse tryptophan hydroxylase 2 (Tph2 or TpH2) promoter/enhancer regions on the BAC transgene. The mhChR2::YFP fusion protein is composed of a mammalian codon-optimized Chlamydomonas reinhardtii-derived channelrhodopsin-2 that was modified to harbor a gain-of-function H134R substitution (mhChR2; also called hChR2-H134R) fused in-frame with an enhanced yellow fluorescent protein (EYFP). The mhChR2 is designed to cause larger stationary photocurrents compared to ChR2. The bacterial opsins are retinal-binding proteins that provide light-dependent ion transport and sensory functions to a family of halophilic bacteria; and this mhChR2 functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating mhChR2-expressing neurons with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing/neural activity in these cells. EYFP expression is visible by direct fluorescence (epifluorescence microscope). TpH2-mhChR2-YFP mice derived from founder line 5 exhibit medium EYFP expression in dorsal raphe nucleus, gigantocellular reticular nucleus, paramedian reticular nucleus, and other serotonergic nuclei. Anti-TpH2 immunofluorescent signal co-localizes specifically with anti-YFP antibody-labeled neurons in the cell types listed above; suggesting mhChR2-EYFP labels all the serotonergic neurons with no ectopic expression. No tissues other than brain were tested for EYFP expression. (J:171571)
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