Intraspinal substance p-containing projections to the sympathetic preganglionic neuropil in pigeon, Columba livia: High-performance liquid chromatography, radioimmunoassay and electron microscopic evidence

Abstract

The present study uses quantitative and electron microscopic methods to investigate the hypothesis that intraspinal substance P-sympathetic preganglionic neuron circuitry exists in vertebrates. 17,18 Radioimmunoassay and high-performance liquid chromatography were used to: (1) characterize the chemical nature of the substance P-like immunoreactivity in the sympathetic preganglionic neuropil; and (2) quantify the relative contributions of brain stem, primary sensory and intraspinal neurons to the substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete thoracic spinal cord transections are also reported. High-performance liquid Chromatographie analyses demonstrate that pigeon substance P-like immunoreactivity co-migrates with synthetic substance P, suggesting that the substance P-like material is authentic substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete preganglionic cell column (inclusive of intermediate spinal laminae V and VII as well as preganglionic neurons located within nucleus intercalatus spinalis); (2) cutting the dorsal rootlets entering the last cervical (C14) and first two thoracic (T1, T2) spinal segments resulted in massive depletion of substance P content in dorsal horn of T1, but no detectable losses within the preganglionic cell column or ventral horn of Tl; and (3) total mid-thoracic (T3–4) spinal cord transection significantly depleted the substance P content in the preganglionic cell column (T3–4) as well as in the dorsal (T1–4) and ventral horns (T2–4). Ultrastructural examination of the sympathetic preganglionic neuropil caudal to spinal transections (survival times of 3–14 days) revealed the presence of numerous, intact, normal appearing substance P-like immunoreactive terminals. Immunolabeled terminals formed asymmetric contacts on medium-sized and small caliber dendrites. Extensive degeneration was evident in this material as well. The ultrastructural features of degenerating processes were distinctive and quite dissimilar in appearance from those exhibiting substance P-like immunoreactive staining. No evidence for damage-induced sequestration of substance P-like material into glial elements was found. The above observations are consistent with earlier findings in rat and pigeon, and provide new quantitative and qualitative evidence to support the hypothesis that intraspinal substance P-containing interneurons contribute to the afferentation to the sympathetic preganglionic neuropil of vertebrates.