Time-dependent alterations in NOS1 immunoreactivity in feline pudendal motoneurons following retrograde axonal transport of diphtheria toxin

Abstract

Neuronal nitric oxide synthase immunoreactivity (NOS1-ir) in sacral somatic motor neurons of normal adult cats was compared with NOS1-ir in cats surviving 1 to 10 weeks after injection of the ADP-ribosylating protein diphtheria toxin (DTX) into one-half of the external anal sphincter. Levels of immunostaining were measured by microdensitometry. In non-operated cats, 60% of motor neurons in the ventrolateral (VL) and Onuf's nucleus (ON) showed high levels of NOS1-ir with lower NOS1-ir in 40%. Intramuscular injection of DTX caused cytopathology in motoneurons in ON, but not in VL with onset at 1 week, and regression by 10 weeks. Immunocytochemistry and microdensitometry disclosed an associated rise in levels of NOS1-ir in both the ipsilateral and contralateral ON at 1 week, which persisted up to 4 weeks, but reduced to normality by 10 weeks. Simultaneous neuronal swelling in ON precluded raised staining intensity being an artifact of neuronal atrophy. Despite restriction of cytopathology to ON, motoneurons in VL also exhibited acute elevation with subsequent normalisation of NOS1-ir over an identical time-course. Conclusions. Since DTX inhibits protein synthesis, (i) activation of NOS1 in acute toxicity probably reflects raised intracellular calcium due to loss of calcium homeostasis; (ii) the bilateral response in ON may indicate uptake of DTX by contralateral pudendal axons crossing the sphincter midline; and (iii) raised NOS1-ir in VL indicates a wider response in nuclei synaptically coupled to ON. Recovery of neuronal morphology and normalisation of NOS1-ir in sublethal toxicity contrast with the protracted elevation of NOS1-ir reported by others following axonal lesions associated with neuronal death and muscle target deprivation.