Short- and mid-term fasting impacts ribosomal protein S6 phosphorylation in zebrafish brain

Fasting triggers metabolic responses in organisms and influences several cellular pathways [1]. There is a growing number of experimental designs addressed to expose living organisms to the fasting condition and evaluate the responses at cellular levels. In the central nervous system (CNS) hypothalamic neurons respond promptly to metabolic signals by phosphorylating proteins and/or synthesizing appetiteregulating neuropeptides. A marker of neuronal response to stimuli, including fasting, is represented by pS6, which corresponds to the phosphorylation of ribosomal protein S6 [2]. The goal of our work is to evaluate the activation of central neurons induced by the short- and mid-term fasting conditions in the model organism Danio rerio, commonly known as zebrafish. Zebrafish is largely used as model in biomedicine as well as in aquaculture field [3]. This study was approved by the Italian Ministry of Health n° 291/2022-PR. Adult fishes (n=10/group – mixed sexes) were divided in three experimental groups: control, four and seven days of fasting. Control group was feed twice/day with SDS (Special Diets Service) 400, a specific aquatic diet for regular maintenance. The four- and seven-day fasting groups were completely food deprived. At the end of the experimental period, animals were euthanized, brains were sampled for western blotting and immunohistochemical analyses. Western blot results revealed a significant increase in phosphorylation of S6 protein in animals exposed to mid-term fasting compared to the control, whereas, surprisingly, the expression decreased in brain homogenates of animals exposed to short time fasting. Immunostaining experiments confirm that the highest immunoreactivity occurred upon seven days of fasting in the zebrafish brain with a strong positivity in the neuronal cells of the dorsal telencephalic areas and in the preoptic area. Less numerous and weakly stained neurons were seen in the hypothalamic area, near the hypothalamic recess. In addition, we detected immunoreactivity to pS6 in the taste buds lining the mouth epithelium in the three experimental groups. pS6 immunopositive cells were identified as sensory cells type, by immunofluorescence stainings. Our data demonstrate that four days of fasting does not result in neuronal activation in zebrafish, while seven days of fasting does. These results may have valuable consequences on the animal welfare, especially in particularly stressful conditions, such as transportation.