VMR in Larvae with Ablated Ventral Diencephalic Otp-Expressing Neurons and DA Neuron Ablation Efficiency, Related to Figure 2
(A) Nitroreductase-mediated ablation of a population of preoptic, posterior tubercular and hypothalamic Otp-expressing neurons in Tg(otpb.A:Gal4)zc67 ; Tg(UAS:Nfsb-mCherry) larvae (control Tg(otpb:Gal4); Tg(UAS:NfsB-mCherry) no metronidazole (5dpf). Dorsal view, anterior at left. Scale bar is 50 μm.
(B) NTR-mediated ablation of opn4a cells in Tg(otpb.A:Gal4)zc67 ; Tg(UAS:Nfsb-mCherry) transgenic larvae (5dpf). Top row shows Tg(otpb:Gal4); (UAS:Nfsb-mCherry) control larvae (no metronidazole), mCherry is co-expressed with a subset of opn4a in the anterior preoptic area (aPO) and posterior tuberculum (PT). Bottom row shows ablated fish. In ablated larvae reduction of opn4a cells in the aPO and PT region is visible (arrowhead). Dorsal view, anterior to the left. Scale bar is 50 μm.
(C) VMR in Tg(otpb.A:Gal4)zc67 ; Tg(UAS:Nfsb-mCherry) neuron ablated larvae (6-7dpf). Ablated larvae (red lines) show reduced VMR compared to controls (black line) (ANOVA; F12,276 =17.42, p < 0.001) (n = 24 larvae). Larval activity was recorded every 10 seconds and averaged over 1 minute intervals. For pairwise comparisons, *p < 0.05, **p < 0.01.
(D) Metronidazole-mediated ablation of posterior tubercular dopaminergic neurons in 7 dpf Tg(BAC th:Gal4VP16)m1233 BAC transgenic line. Dopaminergic neuron groups 2 and 4 are strongly reduced in ablated larvae when compared to control larvae (Tg(BAC th:Gal4VP16) m1233; Tg(UAS: EGFPCAAX)m1230 ) as seen by anti-Tyrosine hydroxylase immunostaining. Groups 3, 5, 6 and 7 dopaminergic neurons, arch associated neurons, area postrema, very few amacrine cells and a subset of Locus coeruleus neurons are also labeled by the transgene and therefore also ablated by using this line. Other TH neurons are not ablated. Dorsal view, anterior to the left. Scale bar is 50 μm.
(E) Quantification of loss of posterior tubercular dopaminergic groups 2 and 4 in metronidazolemediated cell ablation of 7 dpf Tg(BAC th:Gal4VP16) m1233 transgenic line. Control Tg(BAC th:Gal4VP16) m1233; Tg(UAS:EGFPCAAX)m1230 (dark blue, n = 33 larvae) and DA neuron ablated larvae Tg(BAC th:Gal4VP16) m1233; Tg(UAS-E1b:NfsB-mCherry)) (light blue, n = 34 larvae). Quantification reveals a strong reduction of dopaminergic groups 2 and 4 in more than 80% of the ablated larvae. Average percentage and 95% confidence intervals are shown.
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