Figure 1. Comparative expression levels of NPY mRNA in the brain and foregut of young and old turquoise killifish (A) and zebrafish (B). (A) Significant increase in NPY mRNA in the brain of 27 wph animals compared to 5 wph ones. Higher expression levels in the brain of animals at 5 wph compared to the levels detected in the foregut of the same animals. (B) Significant increase in NPY mRNA in the brain of 36-month-old zebrafish compared to 6-month-old animals. Significant decrease in the foregut compared to the brain either in young or old specimens. * (p < 0.5), ** (p < 0.05), *** (p < 0.001).

Figure 2. Age-related changes of NPY immunoreactivity in the brain of zebrafish. (A–C) Transverse sections of the brain of a 6-month-old zebrafish. (A) Overview of NPY immunoreactivity in the hypothalamic area (lateral and dorsal periventricular hypothalamus) and in the midbrain tegmentum of 6-month-old zebrafish. Positive neurons are distributed in the lateral hypothalamus, in the proximity of the preglomerular nucleus and along the ventricle. Abundant varicosities are distributed in the hypothalamus and over the entire tegmentum. (A1) Higher magnification of NPY positive neurons along the ventricle and in the proximity of the preglomerular nucleus. (B) Packed immunoreactive varicose fibers in the semicircular tori. (C) Overview of the optic tectum and the midbrain tegmentum displaying staining in a few neurons of the periventricular grey zone and in varicose fibers over the different layers of the optic tectum, numerous positive neurons along the ventricle and in the proximity of the preglomerular nucleus. (D–E1) Transverse sections of the brain of a 36-month-old zebrafish. (D) Overview of NPY immunoreactivity in the hypothalamic area (lateral and dorsal periventricular hypothalamus) and in the midbrain tegmentum of 36-month-old zebrafish. Few intensely immunostained neurons localized in the lateral and dorsal hypothalamus. Strong immunoreactivity in abundant varicosities distributed in the hypothalamus and projecting fibers toward the optic tectum and over the entire tegmentum. (D1) Higher magnification of projecting fibers toward the optic tectum. (D2) Higher magnification of abundant varicosities distributed in the hypothalamus. (E) Overview of NPY immunoreactivity in fibers in the optic tectum and in varicosities and neurons in the tegmentum. (E1) NPY immunostaining in the optic tectum layers (asterisks) and in a few cells of the periventricular grey zone. Scale bars 100 µm.

Figure 3. NPY immunoreactivity in the foregut of turquoise killifish. (A) Transverse section of the foregut of 5 wph turquoise killifish showing NPY immunopositivity in the cytoplasm of few enteroendocrine cells (arrow). (B) Transverse section of the foregut of 27 wph turquoise killifish showing NPY immunopositivity in the cytoplasm of enteroendocrine cells (arrows) over different villi.

Figure 4. NPY immunoreactivity in the foregut of zebrafish. Transverse sections of the foregut of 6-month-old zebrafish showing NPY immunopositivity in the cytoplasm of enteroendocrine cells (arrows) (A,B) and in varicosities in the myenteric plexus (asterisks) (B).