Projection of non-peptidergic afferents to mouse tooth pulp

A large proportion of pulpal nociceptors are known to contain neuropeptides such as CGRP. However, the projection of non-peptidergic nociceptors to tooth pulp is controversial. Recently, the non- peptidergic subset of nociceptors has been implicated in mechanical pain in the skin. Since mechanical irritation of pulpal nociceptors is critical for evoking tooth pain under pathophysiological conditions, we investigated whether the non-peptidergic afferents project to tooth pulp as potential mechanotransducing afferents. For clear visualization of the non-peptidergic afferents, we took advantage of a recently generated knock-in mouse model in which an axonal tracer, farnesylated green fluorescence protein (GFP), is expressed from the locus of a sensory neuron-specific gene, Mrgprd. In the trigeminal ganglia (TG), we demonstrated that GFP is exclusively expressed in afferents binding to isolectin B4 (IB4), a neurochemical marker of non-peptidergic nociceptors, but is rarely co-localized with CGRP. Retrograde labeling of pulpal afferents demonstrated that a low proportion of pulpal afferents was co-localized with GFP. Immunohistochemical detection of the axonal tracer revealed that GFP-positive afferent terminals were densely projected into the tooth pulp. These results provide convincing evidence that non-peptidergic nociceptors are projected into the tooth pulp and suggest a potential role for these afferents in tooth pain.