Distribution and origins of nitric oxide-producing nerve fibers in the dog tongue: correlated NADPH-diaphorase histochemistry and immunohistochemistry for calcitonin gene-related peptide using light and electron microscopy.

The distribution and origins of nitric oxide (NO)-producing nerves in the dog tongue with reference to calcitonin gene-related peptide (CGRP)-containing sensory fibers were investigated using NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry for CGRP and NO synthase combined with retrograde axonal tracing and denervation experiments. The ultrastructural relationships between NADPH-d-positive and CGRP-immunoreactive neuronal elements were also examined electron microscopically. NADPH-d-positive and CGRP-immunoreactive varicose fibers were found within the taste buds and surrounding the epithelia of the fungiform papillae, and they disappeared completely after severance of the lingual nerve. Following injection of fast blue into the subepithelial layer of the anterior two thirds of the tongue, retrogradely labeled neurons possessing NO synthase and/or CGRP immunoreactivities were mainly detected in the trigeminal ganglion. Some of the retrogradely labeled trigeminal cells showed the coexistence of NADPH-d reactivity and CGRP immunoreactivity, but in the geniculate ganglion neither NADPH-d reactivity nor NO synthase immunoreactivity was found instead of retrogradely labeled CGRP-immunoreactive neurons. The lingual artery and its branches, including the arteriovenous anastomoses, showed dense distributions of NADPH-d-positive fibers, most of which were unaffected by the denervation experiments. There were many small ganglia in the tongue, and virtually all ganglionic neurons were NADPH-d reactive. CGRP-immuno-reactive varicose fibers were also found around the vascular walls and within the intralingual ganglia. Ultrastructural analysis revealed a close distribution of NADPH-d-positive and CGRP-immunoreactive varicose fibers within the arterial walls, and synaptic contacts between CGRP-immunoreactive terminals and NADPH-d-positive intralingual ganglionic neurons. These results indicated that the taste buds of epithelia of fungiform papillae in the anterior two thirds of the dog tongue receive NADPH-d-positive and CGRP-immunoreactive sensory fibers from the trigeminal ganglion, and that perivascular NADPH-d-positive fibers mainly originate from intrinsic ganglia in the tongue. The ultrastructural findings suggest an intrinsic peripheral nerve-reflex mechanism in the regulation of the lingual vascular function by NO-producing postganglionic parasympathetic neurons and CGRP-containing sensory fibers.