(A) As detected by ISH, the immediate early gene c-fos is upregulated in many larval brain areas following A?short injection, including the dorsal and ventral telencephalon (tel) and the posterior hypothalamus (black arrowheads), relative to A?rev control injections. Other upregulated areas in the midbrain and hindbrain are indicated (white arrowheads). hyp- hypothalamus; hb- hindbrain. D = dorsal, p=Posterior, R = Right. n = blind counts of brains with the shown expression pattern/total brains. 24/43 stringently counts only brains with the major areas upregulated. (B) Compared to A?rev injections, A?long oligomers induce less c-fos expression. The A?rev and A?long treated brains were stained longer than in (A) to ensure detection of weaker c-fos expression. n = blind counts of number of brains with the shown expression/total brains. (C) c-fos is upregulated in many larval brain areas at 10 am (ZT1) awake fish, including the dorsal and ventral telencephalon and the posterior hypothalamus (black arrowheads), and other discrete regions of the mid and hindbrain (white arrowheads). c-fos expression is downregulated in later timepoints (ZT13) and is very low in ZT19 brains, when larvae are predominantly asleep. N = 10 fish/timepoint. (D, D?) Brain expression of the neuronal activity correlate pERK/tERK comparing A?short (n = 6) to A?rev (n = 5) injected larvae identified areas upregulated (green) and downregulated (magenta) by A?short. Data are shown as a thresholded maximum projection overlaid on the Z-Brain Atlas tERK reference (gray). White arrowheads indicate regions in the ventral telencephalon and posterior hypothalamus that are upregulated similar to c-fos in (A). Dorsal view in (D), lateral view in (D?). (E, E?) pERK/tERK expression after A?long injections (n = 7) shows widespread downregulation of neuronal activity (magenta) compared to A?rev controls (n = 7), consistent with c-fos data in (B). Dorsal view in (E), lateral view in (E?). (F) As detected by ISH, the number and intensity of hypothalamic galanin-positive neurons are downregulated following A?short injection and upregulated following A?long injection, relative to A?rev control injections. Representative images from N = 22?24 per condition. (G) Normalized, blinded counts of hypothalamic galanin-positive cell numbers 4?6 hr after A?short and A?long injections, relative to A?rev. Error bars indicate ± SEM. The mean difference effect size and 95% confidence interval is plotted at the bottom. **p<0.01, one-way ANOVA. See also Figure 2?source datas 1 and 2.
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