Regional immunohistochemistry of the frog intracardiac ganglia

Abstract

Experimental studies suggest that intracardiac ganglia act as integrative control centers for maintaining the adequate cardiac output. However, the synaptic connections and circuits of intracardiac neurons remain poorly understood. Frog provides a simple model to study the synaptic connections of intracardiac ganglion cells. We employed immunohistochemistry for cholinergic, adrenergic and peptidergic markers to determine regional distribution of the nerve fibers in intact atrial preparations of the frog Rana temporaria. Most frog intracardiac neurons received inputs by 1-3 preganglionic fibers that were positive for choline acetyltransferase (ChAT). In addition, many intracardiac neurons received separate inputs by the fibers that were positive for calcitonin generelated peptide (CGRP). Synaptophysin (SYP)- and ChAT-immunoreactivity co-localized in synaptic boutons on the somata of atrial intracardiac neurons. The neurons in ventricular endocardium had no apparent ChAT- and SYP-positive boutons on their somata. All major intracardiac nerves contained the distinct ChAT-, tyrosine hydroxylase (TH)-, and CGRP-immunoreactive fibers. Dense network of CGRP-positive nerve terminals was found in the venal sinus. The TH-positive fibers enlarged greatly in diameter at the atrio-ventricular junction. Most somata of the intracardiac neurons exhibited ChAT-immunoreactivity, but few TH-positive nerve cells were also found. In spite that some neurons were negative for ChAT, they received ChAT-positive inputs as well. Our findings suggest that (1) most neurons in the intracardiac ganglia of the frog Rana temporaria express cholinergic phenotype, (2) the frog intracardiac neurons receive both cholinergic and peptidergic inputs, and (3) distribution patterns of the adrenergic and peptidergic fibers exhibit regional-specific heterogeneity in the frog heart.