Distinct subsets of neuropeptide Y-negative principal neurons receive basket-like innervation from enkephalinergic and gabaergic axons in the superior cervical ganglion of adult rats.

The distributions of axons immunoreactive for [Leu]- or [Met]enkephalin and GABA were studied in the superior cervical ganglion of adult rats. The antigens were visualized separately and in combination with neuropeptide Y by the immunoperoxidase technique, using reaction end-products of different colors. Similarities and differences were found in the light-microscopic innervation patterns of enkephalin- and GABA-immunoreactive nerve fibers. Both fiber systems were heterogeneously distributed within the superior cervical ganglion, forming denser networks in its rostral part than elsewhere in the ganglion. The appearance of labeled nerve fibers differed in the two systems. Enkephalin-immunoreactive axons exhibited dotted profiles due to a strong immunoreaction in the axonal varicosities as compared with that in the intervaricose segments, whereas GABA-positive fibers were evenly labeled in both parts of the axons. The most marked difference between the innervation patterns from enkephalin- and GABA-immunoreactive axons was the presence of bundles of varicose axons in conjunction with the basket-like aggregation of enkephalin-immunoreactive nerve terminals. The possibility that enkephalins and GABA are co-localized in certain axons was excluded in double-labeling studies, silver intensification being used for the first antigen and the nickel-enhanced diaminobenzidine reaction for the second antigen. Different subsets of principal neurons were richly innervated in a basket-like manner by axons immunoreactive for enkephalins and GABA. Additionally, combined staining with antisera against either enkephalin and neuropeptide Y or GABA and neuropeptide Y revealed that both subsets of principal neurons richly innervated either by enkephalin-immunoreactive or by GABA-immunoreactive axons were devoid of neuropeptide Y immunoreactivity. Thus, the enkephalinergic and GABAergic axons have different subpopulations of neuropeptide Y-negative principal neurons as targets in the superior cervical ganglion. These results provide further evidence that sympathetic ganglion cells can be classified on the basis of their receiving input from different sources.