Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus

Abstract

A constitutive NOS complementary DNA (cDNA) was partially cloned by RT-PCR from the brain of a teleost, the Nile tilapia (Oreochromis niloticus), using degenerate primers against conserved regions of NOS. The predicted 206-long amino acid sequence showed a high degree of identity with other vertebrate neuronal NOS (nNOS) protein sequences. In addition, phylogenetic analysis revealed that Nile tilapia NOS clustered with other known nNOS.

Using the coupled reaction of semi-quantitative RT-PCR and Southern blotting, the basal tissue expression pattern of the cloned nNOS gene was investigated in discrete areas of the central nervous system (CNS) and in the heart and skeletal muscle tissue. As revealed, expression of nNOS transcripts was detected in all the CNS regions examined, whereas nNOS gene was not expressed in the heart and skeletal muscle. The distribution pattern of nNOS gene expression showed the highest expression levels in the forebrain followed by the optic tectum, the brainstem and the spinal cord, whereas scarce expression was detected in the cerebellum.

Cellular expression of nNOS mRNA was analyzed in the CNS by means of in situ hybridization. According to the RT-PCR results, most nNOS mRNA expressing neurons are localized in the telencephalon and diencephalon, whereas in the mesencephalic optic tectum, the brainstem and the spinal cord, nNOS mRNA expressing neurons are relatively more scattered. A very low hybridization signal was detected in the cerebellar cortex.

These results suggest that NO is involved in numerous brain functions in teleosts.