Abstract
Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.