Transgene expression of the mhChR2::YFP fusion protein is directed to cholinergic neuronal populations by the mouse choline acetyltransferase (Chat or ChAT) 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 BAC used to generate the construct includes the vesicular acetylcholine transporter gene (Slc18a3 or VAChT). Slc18a3 is located in the intron between the first and second exons of the Chat gene and is under the control of the Chat promoter. VAChT protein is overexpressed in the hippocampus and brainstem. 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). ChAT-mhChR2-YFP mice derived from founder line 6 exhibit strong EYFP expression in striatum, trochlear nucleus, medial habenula, interpeduncular nucleus and various brainstem motor neuron nuclei. Lower EYFP expression is found in cortex, hippocampus, and other brain regions. ChAT co-staining shows precise co-localization with mhChR2-EYFP expression neurons; suggesting mhChR2-EYFP labeled neurons in this line are cholinergic neurons. High EYFP fluorescence is also observed in the ventral gray horn in transverse section of the spinal cord. The donating investigator also reports that these ChAT-mhChR2-YFP line 6 mice exhibit a similar expression pattern as ChAT-mhChR2-YFP line 5 (JAX Stock No. 014545), but line 6 mice have brighter EYFP expression. (J:171569, J:197166)
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