GnRH3 neuronal ablation is validated in the brain at 28 days of treatment. (A) Treatment and analysis schedule. Fish were exposed to 2 mM Mtz daily during the dark phase for 28 days, and were sampled at 14 and 28 days into the treatment. (B) Top panel: confocal images of GnRH3 neurons expressing NTR-mCherry fusion protein (magenta) in whole brains from the Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) fish before the treatment, and at 14 and 28 days of treatment. Bottom panel: POA-zoomed images showing the boxed area from the top images. Scale bars, 200 µm. (C) Confocal images of co-immunostaining of GnRH3-associated peptide (green) and mCherry (magenta) in the whole brain of Tg-Mtz (top panels) and Tg-Cont (bottom panels) at 28 days of treatment. GnRH3 neuron cell bodies co-localized with mCherry in Tg-Mtz are labeled by arrowheads. Scale bars, 100 µm. Insets, zoomed images of GnRH3-ir and mCherry-ir neuron cell bodies co-localization showing the boxed areas of the merged image in Tg-Mtz. Scale bars, 10 µm. (D) Numbers of GnRH3-positive cells (left) and mCherry-positive cells (right) in the POA (n = 6–7). (E) Relative gnrh3 mRNA levels in the whole brain (n = 4 each). OB, olfactory bulb; TL, telencephalon; POA, preoptic area; ir, immunoreactive; WT-Mtz, WT siblings treated with 2 mM metronidazole (Mtz); Tg-Cont, Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) treated with vehicle; Tg-Mtz, Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) treated with 2 mM Mtz. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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