Nitric oxide synthetase (NOS)‐containing sympathoadrenal cholinergic neurons of the rat IML‐cell column: Evidence from histochemistry, immunohistochemistry, and retrograde labeling

Nitric oxide synthetase (NOS) can be selectively stained in neurons by either NAJIPHdiaphorase (i.e., NOS)-histochemistry or immunohistochemistry with antibodies raised against NOS, which apparently label identical reactive sites (Hope, B.T., G.J. Michael, K.M. Knigge, and S.R. Vincent, Proc. Natl. Acad. Sci. USA 88:2811-2814, '91). We provide histochemical evidence for the existence of a neuron-specific NOS-activity in autonomic neurons of the thoracic spinal cord. Among the four main preganglionic cell clusters investigated at midthoracic levels, Th7-10, the intermediolateral (1ML)-cell column was the most prominently stained cell group. The histochemical staining was absent in other spinal cord neurons and non-neuronal cells, e.g., GFAP-positive glial cells. Staining was completely blocked by ""-nitroL-arginine (L-"A), a potent NOS-inhibitor for brain and peripheral autonomic neurons, but was still observed in the presence of another NOS-inhibitor, Nu-monomethyl-L-arginine (MeArg). The NOS-activity co-localized with nearly half of the ChAT-immunostained neurons located in the mid-thoracic IML-cell column as quantified by cell counts in single and double-stained tissue sections. We conclude that NOS-activity-containing neurons represent a distinct group among cholinergic IML-neurons, which suggests a more general function of this newly defined subpopulation of the spinal cord autonomic system. In vivo Fast blue retrograde labeling combined with histochemical staining and immunostaining revealed that sympathoadrenal projection neurons belong to the distinct NOS and ChAT-positive IML-cell group. The results are consistent with the idea of a crucial role of NOS and the related L-argininedependent nitric oxide biosynthesis for adrenomedullary innervation and autonomic control of adrenal gland function. In the adult rat, the intermediate region of the thoracic spinal cord (comprising laminae VI, VII, and X according to the classification of Rexed, '54) contains sympathetic preganglionic neurons (SPNs) that form distinct cholinergic cell clusters (Navaratnam and Lewis, '70; Borges and Iversen, '86) and are referred to as the central autonomic (CA),