Astyanax mexicanus is a teleost fish model used for evolution studies. Among the same species, there are several populations of sighted surface fish (SF) that live in Mexican rivers and at least twenty nine populations of blind cavefish (CF) that live in perpetual darkness. Several of these cave populations are independently-evolved, and they derived from surface fish-like ancestors. CF have lost their eyes and their pigmentation, and they have also evolved a number of behavioral traits. Most CF populations have lost the aggressive behavior that is a trademark of their SF counterparts. Here we characterized behavioural differences between SF and CF and we investigated the modifications of neural networks responsible for the loss of aggressiveness in cavefish. We first characterized aggressive behavior in Astyanax. Using an “intruder assay”, we found that SF not only attack ten times more during a one hour period, but also show a significantly different pattern in the temporal distribution of their attacks: while two CF attack mostly during the first minutes, SF attack more and more frequently as time goes by during the test. Then we demonstrated that the loss of aggressiveness in CF is not due to their blind phenotype, and using hybrids and independently-evolved populations of CF we could suggest that aggressive behavior in SF is genetically-encoded. After pharmacological experiments using compounds modulating serotonin levels or using SF and CF receiving different food regimes, we hypothesized that the loss of aggressiveness of CF may correspond to an adaptation to cave life, as they spend most of their time looking for food. On the other hand, the aggressiveness of SF is closely connected to the hierarchy within the school, dominance itself being mainly due to the levels of serotonin in the raphe. Thus, serotonin levels are inversely correlated with agonistic behavior. Neuroanatomical analyses on SF and CF brains showed an identical organization of their serotonin neuronal networks, but one of the serotonergic hypothalamic nucleus was significantly larger in CF and contained more neurons. Treatments of embryos with cyclopamine, an inhibitor of Sonic hedgehog (Shh) signaling, showed that this enlargement is induced by the Shh signaling pathway, itself known to be amplified during the development of the CF. In fact, the serotonergic system is not only one that is changed in the nervous system of CF. HPLC measurements showed that all aminergic systems are amplified in CF, which show a sort of “hyper-aminergic” phenotype. Studies comparing the neuroanatomy of aminergic systems and the activity of amine-degrading enzymes between SF and CF showed that variations in both the number of neurons and the activity of degrading enzymes converge towards an amplification of aminergic neurotransmission and are responsible for the phenotype. In parallel, we established transgenesis methods in Astyanax. We showed that techniques using meganuclease or transposons are valuable with our fish species to generate transgenic lines. This tool will be used to test the importance and function of genes of interest in the development of the nervous system and associated behaviors.
