Neuropeptide-Y gene expression in the goldfish brain: distribution and regulation by ovarian steroids

Abstract

Neuropeptide-Y (NPY) has been recently cloned from a goldfish cDNA library. Using a probe derived from this cDNA clone, the distribution and regulation of NPY mRNA in the goldfish brain were examined in the present study by in situ hybridization, Northern blot analysis, and ribonuclease protection assay. Frozen tissue sections from the goldfish brain and pituitary were hybridized with a digoxigenin-labeled RNA probe. Hybridization signal was detected mainly in fore-brain regions, particularly in the nucleus entopeduncularis of the ventral telencephalon, the preoptic area (POA), the olfactory bulbs, and various thalamic regions. In the midbrain, NPY mRNA was found in the optic-tectum and locus coeruleus. Northern blot hybridization of total RNA extracted from different brain areas with a 32P-labeled RNA probe detected a single mRNA species and confirmed that most NPY mRNA was present in telencephalon/POA and optic-tectum/thalamus. Our previous physiological studies showed that the ovarian steroids testosterone (T) and estradiol (E2) have potentiating effects on the actions of NPY on gonadotropin and GH release. Therefore, we also tested the possibility that T and E2 may modulate NPY gene expression. Fish were implanted ip with pellets containing T, E2, or no steroid (control) for 5 days, RNA was extracted, and NPY mRNA levels were estimated using a ribonuclease protection assay. Pretreatment with T or E2 induced a 2- to 3-fold increase in NPY mRNA levels in the telencephalon/POA, but not in the optic-tectum/thalamus. In situ hybridization using brains taken from T-implanted fish demonstrated that the site of steroid action is the POA. This report represents the first study on NPY gene expression in a nonmammalian species and demonstrates that 1) NPY mRNA is present in the neuroendocrine regulatory centers; and 2) ovarian steroids stimulate NPY gene expression in the POA.