Orexin as an input of circadian system in goldfish: Effects on clock gene expression and locomotor activity rhythms

Orexins are neuropeptides mainly known for regulating feeding behavior and sleep-wakefulness cycle in vertebrates. Daily variations of orexin-A expression have been reported in fish, with the highest levels preceding feeding time. However, it is unknown if such variations could be related with daily rhythms of clock genes, which form the molecular core of circadian oscillators. The aim of the present study was to identify the possible role of orexin as an input element of the goldfish circadian system. It was investigated the effects of orexin-A (10ng/gbw) intracerebroventricular injections on the expression of clock genes, NPY and ghrelin, as well as on daily locomotor activity rhythms. Goldfish held under 12L:12D photoperiod and injected at midday with orexin or saline, were sacrificed at 1 and 3h post-injection. The analysis of genes expression by qReal Time PCR showed an increment of Per genes in hypothalamus and foregut at 3h post-injection, but not in hindgut and liver. The gBmal1a expression remained unaltered in all the studied tissues. Orexin induced NPY in the hypothalamus and ghrelin in the foregut. Locomotor activity was studied in fish daily injected with orexin for several consecutive days under different experimental conditions. Orexin synchronized locomotor activity in goldfish maintained in 24L and fasting conditions. Present results support a cross-talking between orexin-A and other feeding regulators at central and peripheral level, and suggest, for the first time, a role of this peptide as an input of the circadian system in fish.