Developmental and Regional Expression of NADPH-diaphorase/nitric Oxide Synthase in Spinal Cord Neurons Correlates with the Emergence of Limb Motor Networks in Metamorphosing Xenopus Laevis

Metamorphosis in anuran amphibians requires a complete transformation in locomotor strategy from undulatory tadpole swimming to adult quadrupedal propulsion. The underlying reconfiguration of spinal networks may be influenced by various neuromodulators including nitric oxide, which is known to play an important role in CNS development and plasticity in diverse species, including metamorphosis of amphibians. Using NADPH-diaphorase (NADPH-d) staining and neuronal nitric oxide synthase (nNOS) immunofluorescence labelling, the expression and developmental distribution of NOS-containing neurons in the spinal cord and brainstem were analysed in all metamorphic stages of Xenopus laevis. Wholemount preparations of the spinal cord from early stages of metamorphosis (coincident with emergence of the fore- and hindlimb buds) revealed two clusters of NOS-positive neurons interspersed with areas devoid of stained somata. These cells were distributed in three topographic subgroups, the most ventral of which had axonal projections that crossed the ventral commissure. Motoneurons innervating the fore- and hindlimb buds were retrogradely labelled with horseradish peroxidase (HRP) to determine their position in relation to the two NOS-expressing cord regions. Limb motoneurons and NOS-positive cells did not overlap, indicating that during early stages of metamorphosis nitrergic neurons are excluded from regions where spinal limb circuits are forming. As metamorphosis progresses, NOS expression became distributed along the length of the spinal cord together with an increase in the number and intensity of labelled cells and fibers. NOS expression reached a peak as the forelimbs emerge then declined. These findings are consistent with a role for nitric oxide (NO) in the developmental transition from undulatory swimming to quadrupedal locomotion.