Nitric oxide synthase and the contribution of nitric oxide to vertebrate motor control

I have explored the role of nitric oxide (NO) and the distribution of its synthetic enzyme in neurons, nNOS, in vertebrate motor control. The CNS of Xenopus laevis tadpoles was used primarily in this investigation as a developmental model of neural plasticity. During metamorphosis, spinal locomotor networks for axial‐based swimming in the tadpole undergo dynamic reconfiguration in order to generate mature rhythmic motor patterns for limb‐based propulsion in the frog. Thyroid hormones (THs) orchestrate this change, possibly involving NO signalling in the CNS. Anatomical data were obtained from organotypic brainstem‐spinal cord cultures using histochemical techniques. THs induced NOS expression in the spinal cord of stage 47, premetamorphic CNSs, a developmental stage when NOS is normally virtually absent in this region, after 3 days of organotypic culture. The pattern of NOS‐expressing cells was found to be similar to that of more mature tadpoles, providing evidence that one developmental effect of THs in Xenopus CNS is to induce the expression of the NOS enzyme in a regionally specific manner. The role of NO on the maturation of motor systems in the spinal cord was subsequently explored further, again using organotypic culture methods. Xenopus CNSs at stage 47 were cultured in the presence of the NO donor SNAP, or the NOS inhibitor L‐NAME. Primary motor neurons in the spinal cord serve embryonic swimming; secondary motor neuron pools provide motor innervation to the limbs, but also to mature larval and adult axial musculature. During development, secondary motor systems eventually supersede the primary system. In my organotypic cultures at stage 47, L‐NAME had no significant effect, but exogenous NO was found to cause an increase in the number of presumptive secondary motor neurons in the brachial region of the spinal cord, as indicated using choline acetyltransferase (ChAT) immunohistochemistry. This indicates that NO has a differentiating effect on secondary motor neurons in this region and provides evidence suggesting a maturational role of NO in the reconfiguration of spinal locomotor circuitry. Finally, the presence of NOS in the spinal cord of the lamprey, a primitive vertebrate, was explored using histochemical techniques. The lamprey spinal cord is a well‐studied model of rhythm generation by CPGs. NOS expression was found in motor and sensory cells involved in the generation and modulation of locomotion: motor neurons, dorsal cells, edge cells, and giant interneurons labelled for both NADPH‐diaphorase and nNOS. This suggests that NO might exert a modulatory influence on rhythmic locomotion in the lamprey. Furthermore, my data indicate NO’s role in motor control appeared early in vertebrate evolution, and that NO signalling in the CNS has been evolutionarily conserved. Taken together, data from Xenopus and lamprey suggest an important role for NO in the development and execution of vertebrate motor control

Nitric oxide modulation of the electrically excitable skin of <em>Xenopus laevis</em> frog tadpoles

Nitric oxide (NO) is a highly diffusible signalling molecule with widespread effects on the integrative electrical properties of a variety of neuronal and muscle cells. We have explored the effects of NO on the cardiac-like impulse generated by skin cells of the hatchling Xenopus tadpole. Skin cell impulses propagate from cell to cell via gap junctions and form an unusual sensory system, which triggers escape behaviour at early stages of amphibian development. We show that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increases the duration of the skin impulse and slows the rate of impulse propagation across the skin, and also produces a significant depolarization of the membrane potential of skin cells. Each of these effects of SNAP is significantly reversed by the NO scavenger, C-PTIO. Possible sources of NO have been investigated using both NADPH-diaphorase histochemistry and nNOS immunocytochemistry to label the enzyme nitric oxide synthase (NOS), and DAF-2 to label NO itself. In each case a punctate distribution of skin cells is labelled, indicating that the endogenous production of NO may regulate the properties of the skin impulse.</p