Representative photomicrographs illustrating increased colocalization of transcripts of the immediate early gene cfos, a marker of neuronal activation, within hcrt neurons after optogenetic activation with blue light exposure in the brains of transgenic Hcrt:ChR2-EYFP54 larval zebrafish (6 dpf) using RNAscope staining. (a) Representative confocal photomicrograph (×25) illustrates the 6 dpf zebrafish brain in a dorsal/ventral view after 20-min of red-light exposure, a wavelength of light that fails to optogenetically activate Hcrt neurons. Brains were counterstained by DAPI (blue) and labeled for hcrt (green) and cfos (magenta), with merged photos showing an overlay of each channel and boxes showing single-cell enlargements of DAPI (Box 1), hcrt (Box 2), cfos (Box 3), and the merge of each channel (Box 4) showing a weak colocalization between cfos and hcrt. (b) Representative confocal photomicrograph (×25) illustrates the 6 dpf zebrafish brain in a dorsal/ventral view after 30-min of blue-light exposure, a wavelength of light that optogenetically activates Hcrt neurons. Brains were counterstained by DAPI (blue) and labeled for hcrt (green) and cfos (magenta), with merged photos showing an overlay of each channel and boxes showing single-cell enlargements of DAPI (Box 5), hcrt (Box 6), cfos (Box 7), and the merge of each channel (Box 8) showing strong colocalization between cfos and hcrt. Scale bars: low-magnification, 10 µm, high-magnification, 2 µm. dpf, days post fertilization.
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