Origin of Neuronal Nitric Oxide Synthase (NOS)-Immunoreactive Fibers in Guinea Pig Parasympathetic Cardiac Ganglia MICHELLE A. CALUPCA, 1 MARGARET A. VIZZARD, 2 AND RODNEY L. PARSONS 1 * 1 Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, Vermont 05405 2 Department of Neurology, College of Medicine, University of Vermont, Burlington, Vermont 05405 ABSTRACT This study was conducted to determine the origin(s) of neuronal nitric oxide synthase- immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations contain- ing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Com- parison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1–5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia. J. Comp. Neurol. 426: 493–504, 2000. © 2000 Wiley-Liss, Inc. Indexing terms: NOS; vagal afferent fibers; nodose The vertebrate parasympathetic cardiac ganglia, once thought to be simple relay stations, are now recognized as potential integration centers regulating the parasympa- thetic inhibitory drive to cardiac tissues (Parsons et al., 1987; Konopka et al., 1992; Randall and Wurster, 1994). Postganglionic parasympathetic cardiac ganglia neurons receive sensory and postganglionic sympathetic fiber in- puts as well as parasympathetic preganglionic inputs (Hardwick et al., 1995; Kennedy et al., 1998). Both spinal and vagal afferent fibers contribute to the input to the heart and thus could be the source of afferent fibers present within the cardiac ganglia (Dalsgaard et al., 1986; Quigg, 1991). Spinal afferent fibers derived from lower cervical to upper thoracic dorsal root ganglia (DRG), which innervate the heart, exhibit both substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreac- tivity (Urban and Papka, 1985; Dalsgaard et al., 1986; Gibbins et al., 1987). SP-/CGRP-immunoreactive (IR) fi- Grant sponsor: National Institutes of Health; Grant numbers: NS 23978 and DK 51369. *Correspondence to: Rodney L. Parsons, PhD, Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail:rparsons@zoo.uvm.edu Received 11 May 2000; Revised 11 July 2000; Accepted 11 July 2000 THE JOURNAL OF COMPARATIVE NEUROLOGY 426:493–504 (2000) © 2000 WILEY-LISS, INC.