Embryonic Ethanol Exposure Affects the Early Development, Migration, and Location of Hypocretin/Orexin Neurons in Zebrafish

Background Embryonic ethanol (EtOH) exposure is known to increase alcohol drinking later in life and have long‐term effects on neurochemical systems in the brain. With zebrafish having marked advantages for elucidating neural mechanisms underlying brain disorders, we recently tested and showed in these fish, similar to rodents, that low‐dose embryonic EtOH stimulates voluntary consumption of EtOH while increasing expression of hypocretin/orexin (hcrt) neurons, a neuropeptide that promotes consummatory and reward‐related behaviors. The goal of the present study was to characterize how embryonic EtOH affects early development of the hcrt system and produces persistent changes at older ages that may contribute to this increase in EtOH consumption. Methods We utilized live imaging and Imaris software to investigate how low‐dose embryonic EtOH (0.5%), administered from 22 to 24 hours postfertilization, affects specific properties of hcrt neurons in hcrt:EGFP transgenic zebrafish at different ages. Results Time‐lapse imaging from 24 to 28 hpf showed that embryonic EtOH increased the number of hcrt neurons, reduced the speed, straightness, and displacement of their migratory paths, and altered their direction early in development. At older ages up to 6 dpf, the embryonic EtOH‐induced increase in hcrt neurons was persistent, and the neurons became more widely dispersed. These effects of embryonic EtOH were found to be asymmetric, occurring predominantly on the left side of the brain, and at 6 dpf, they resulted in marked changes in the anatomical location of the hcrt neurons, with some detected outside their normal position in the anterior hypothalamus again primarily on the left side. Conclusions Our findings demonstrate that low‐dose embryonic EtOH has diverse, persistent, and asymmetric effects on the early development of hypothalamic hcrt neurons, which lead to abnormalities in their ultimate location that may contribute to behavioral disturbances, including an increase in EtOH consumption. Here we performed live imaging of hcrt:EGFP zebrafish to evaluate how embryonic exposure to ethanol at a low concentration affects the early development of hypocretin/orexin (hcrt) neurons, a neuropeptide that promotes consummatory and reward‐related behaviors. Ethanol increased the number of hcrt neurons, altered their migration, and increased their dispersion. These effects were asymmetric, mostly on the left side of the brain, and caused hcrt neurons to become abnormally