Immunohistochemical distribution and localization of neuronal nitric oxide synthase in brain and retina of Epinephelus coioides

【Objective】The content of nitric oxide（NO），distribution and activity of neuronal nitric oxide synthase （nNOS）in brain and retina of Epinephelus coioides were studied to provide foundation for further revealing the mechanism of action of NO in the nervous system of E. coioides.【Method】NADPH-d staining，immunohistochemical method， western blotting and Griess reaction were used to study the distribution，activity of nNOS and NO content in the brain and retina.【Result】The positive material of nitric oxide synthase（NOS）was stained blue using NADPH-d method. NOS positive materials were found in neurons and nerve fibers in the cerebrum. In the cerebellum，NOS was distributed in granular cells of the granular layer，nerve fibers of the molecular layer，and Purkinje cells of the Purkinje cell layer. In the retina， the NOS was found to be distributed in the pigment epithelium layer，rod and cone layer，outer nuclear layer，outer plexiform layer，inner nuclear layer，inner plexiform layer，ganglion cell layer and retinal nerve fiber layer. In addition，the immunohistochemical positive materials of nNOS were stained dark brown. In the cerebrum，nNOS was distributed in neurons and nerve fibers. In the cerebellum，nNOS was distributed in the granular layer，molecular layer and Purkinje cell layer. In the retina，nNOS was found to be distributed in the pigment epithelium layer，rod and cone layer，outer nuclear layer，outer plexiform layer，inner nuclear layer，inner plexiform layer，ganglion cell layer and retinal nerve fiber layer. The result of western blotting showed that there were three protein blotting bands on the nitrocellulose membrane after color development for nNOS in the brain and retina，their corresponding molecular weights were 80，120 and 130 kD，respectively. The NO content in brain and retina were 17.601±1.743 and 13.624±1.249 mol/L，respectively. The nNOS activity in brain and retina were 38.070±3.047 and 23.748±2.860 U/mg，respectively.【Conclusion】nNOS exists in brain and retina of E. coioides，and it is speculated that nNOS may play an important role in the cranial and visual nervous system physiological activity of fish. The NO content and nNOS activity in brain are significantly higher than those in retina，presumably because the brain is the important central nervous system and plays an important role in regulating the physiological functions of body.