Developmental changes of NADPH-diaphorase positive structures in the isthmic nuclei of the chick

Nicotine adenine dinucleotide phosphate-diaphorase staining was used to study nitric oxide synthase activity and distribution in the midbrain visual structures of white leghorn chick embryos and post-hatched chickens. Enzyme staining first appeared in the isthmic region at the tenth embryonic day (E10) in the neuropil of the nucleus isthmi, pars parvocellularis. At E11 faint enzyme positivity appeared also in the nucl. isthmi pars magnocellularis, the nucl. semilunaris and the nucl. isthmo-opticus. The staining intensity of the isthmic nuclei dramatically increased between the 12.5th and the 13th days of incubation. The nucl. isthmi, pars parvocellularis showed the strongest enzyme reaction throughout embryonic life. A day before hatching all the isthmic nuclei were heavily stained, however, nicotine adenine dinucleotide phosphate-diaphorase-positive cells occurred exclusively in the nucleus isthmo-opticus. In the tectum opticum, intensely stained cells occupied the stratum fibrosum et griseum superficiale. The layer containing the projection neurons to the isthmo-optic nucleus was unstained. In the isthmic region, the intensity of staining surpassed that of the tectum and reached its maximum at E17 and then slowly decreased till the end of the experimental period (120 days post-hatched). The tractus isthmo-opticus showed nicotine adenine dinucleotide phosphate-diaphorase activity throughout the investigated period of life of the chicken, but the tractus tectoisthmo-opticus was unstained. Our results suggest that in the isthmic nuclei, nicotine adenine dinucleotide phosphate-diaphorase-positive neurons occur only in the isthmo-optic nucleus and optic tectum. The other positively stained structures are the fibers and terminals of tectal cells. In most brain areas nicotine adenine dinucleotide phosphate-diaphorase indicates nitric oxide synthase that produces nitric oxide. The transient appearance of this molecule is probably necessary for neuronal differentiation or the establishment of synaptic connections in the isthmic nuclei, and these developmental changes are under the control of the optic tectum.