A quantitative study of the neuronal nitric oxide synthase expression in the superficial layers of the adult rat superior colliculus after perinatal enucleation.

The level of expression of the neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of rat superior colliculus (SC) was investigated in adult rats after neonatal enucleation using two biochemical methods: (1) measurement of the in vitro specific-activity of NOS by the conversion of [3H]-arginine to [3H]-citrulline and (2) immunochemical analysis by western blotting and densitometry of immunoreactive bands using antibodies that recognise the three prominent isoforms of nNOS, alpha, beta and gamma. A total of 20 Lister rats were used in this study. We have shown that the deprivation of the retinocollicular projections at early postnatal ages induces no significant change in the specific-activity of nNOS. We also have shown that the deafferentation does not significantly influence either the total amount of nNOS in the SC superficial layers or the relative contribution ratio of nNOS isoforms. In conclusion, the expression and activity of nNOS in the SC retinorecipient layers was shown not to be dependent on the presence of retinal afferents during development.

Nitrergic dendrites in the superficial layers of the rat superior colliculus: retinal afferents and alternatively spliced isoforms in normal and deafferented animals.

The superficial layers of the rat superior colliculus (sSC) receive innervation from the retina and include nitrergic neurons. We have shown previously that in sSC, eye enucleation reduces NADPH diaphorase staining considerably in all but the most proximal dendrites of nitrergic neurons. We have used immunocytochemistry for neuronal nitric oxide synthase (nNOS) at light and electron microscopic levels and bilateral eye enucleation with varied survival times to determine the regulatory changes imposed by the direct and indirect loss of retinal input on apparent nNOS amount and subcellular distribution. In addition, we have used SDS-PAGE and immunoblotting to test alternatively spliced isoforms in normal and deafferented animals. Our results show that unambiguously identified retinal terminals contact nitrergic neurons. In normal dendrites, nNOS immunoreactivity was distributed almost completely within the cytoplasm of the dendrite and along the postsynaptic membrane at synaptic junctions, in association with endoplasmic reticulum, ribosomes and external mitochondrial membranes. In contrast, nNOS labeling was greatly reduced in sSC deprived of retinal projections, and could only be observed in association with mitochondrial membranes and postsynaptic densities. Immunoblots of the soluble fraction from sSC revealed a surprisingly high proportion of the beta isoform with respect to the alpha counterpart in normal colliculi, suggesting an increase in isoform proportion after enucleation, or at least maintenance of the same proportion. It is suggested that ultrastructural alterations observed in sSC cells of enucleated animals may be consequent to plastic reactions of the sSC cells in response to the removal of retinal afferents.