Immunolocalization of nitric oxide synthases at the postsynaptic domain of human and rat neuromuscular junctions--light and electron microscopic studies.

Abstract

Neuronal (n) and inducible (i) nitric oxide synthase (NOS) were immunolocalized at the postsynaptic domain of human and rat neuromuscular junctions (NMJs) by light and electron microscopy. We applied polyclonal and monoclonal antibodies for colocalization with three other synaptic proteins, utilizing double and triple fluorescence labeling, and gold and peroxidase for immunoelectron microscopy. By light microscopy, nNOS and iNOS colocalized with desmin and dystrophin, known postsynaptic components, but not with neurofilament protein, a presynaptic component. By electronmicroscopy, nNOS, but not iNOS, colocalized postsynaptically on the same structures as desmin; iNOS was also postsynaptic, but did not colocalize with desmin immunoreactivity. At the NMJs of Duchenne muscular dystrophy patients, both nNOS and iNOS were strongly immunoreactive. At the NMJs of a patient with myasthenia gravis, nNOS was weaker than in controls. Total denervation of rat sciatic nerve did not cause any decrease of nNOS or iNOS immunoreactivity 7 days thereafter. At 15 days after denervation, there was a gradual decrease of immunoreactivity, and immunoreactivity disappeared 30 days after denervation, corresponding to the ultrastructurally detectable disorganization of the postsynaptic region. This seems to be the first combined light and electron microscopic description of the postsynaptic localization of nNOS and iNOS at human and rat NMJs.