Nitrergic and peptidergic innervation in the developing rat heart

The phenotypic expression and anatomic distribution of nitrergic and peptidergic innervation in the developing rat heart was localized by reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and immunohistochemistry using antibodies against neuronal isoform of nitric oxide synthase (nNOS), neuropeptide Y (NPY) and calcitonin-gene-related peptide (CGRP). NPY-immunoreactive nerve fibers showed the earliest expression by 16 days of gestation, with preferential innervation of the nodal and perinodal areas, followed by the innervation of the valves and ventricles by postnatal day 7. NPY immunoreactivity was also localized to a large proportion of the intrinsic cardiac ganglia from 16 days of gestation onwards with a progressive increase in the number of neuronal cell bodies per ganglia with age. CGRP-positive nerve fibers appeared by 19 days of gestation and were less dense during the gestational and early postnatal periods, and showed a quantitative increase in density by 7 days, followed by a decrease by 3 weeks postnatal. None of the intrinsic ganglia were stained positive for CGRP, indicating the extrinsic sensory origin of these stained fibers. Nitrergic innervation paralleled the sensory innervation, with the cardiac ganglia and nerve fibers showing a positive labeling from 19 days of gestation onwards. NADPH-d and nNOS were partially co-localized. Double-label immunohistochemistry showed that a considerable proportion of sensory CGRP-immunopositive fibers were also immunoreactive for NOS. The results of the present study show that neuropeptides and nitric oxide are expressed by the late gestational period and that autonomic efferent innervation precedes sensory and nitrergic innervation in the developing heart.