There exists very little information on the ultrastructure of substance P immunopositive (+) fibers in the human dental pulp, which may help in understanding the mechanism for substance P associated pulpal inflammatory pain. To address this issue, we investigated the presence of substance P+ fibers in the human dental pulp by light- and electron-microscopic immunohistochemistry. Light microscopy revealed that substance P+ fibers ran within neurovascular bundles in the radicular pulp and in the core of coronal pulp. They were also frequently present in the peripheral pulp. Substance P+ fibers showed beads like swellings interconnected by thin axonal strand, in a manner similar to bouton en passants and interconnecting axonal strand in the spinal cord. Electron microscopy revealed that almost all the substance P+ axons were unmyelinated. The axonal swellings of the substance P+ contained numerous clear round vesicles (40-50 nm in diameter) and many large dense-cored vesicles (80-110 nm in diameter) as well as many mitochondria. The vesicles and mitochondria were rarely observed in the thin axonal strand interconnecting the swellings. Intimate interrelationship or synaptic structure between the swellings of substance P+ axon and nearby pulpal cells or axons was not found. These findings suggest co-release of substance P and glutamate from the substance P+ pulpal axons and its action on nearby structures in a paracrine manner.

The ultrastructural parameters related to synaptic release of endings which are presynaptic to tooth pulp afferent terminals (p-endings) were analyzed to understand the underlying mechanism for presynaptic modulation of tooth pulp afferents. Tooth pulp afferents were labelled by applying wheat-germ agglutinin conjugated horseradish peroxidase to the rat right lower incisor, whereafter electron microscopic morphometric analysis with serial section and reconstruction of p-endings in the trigeminal oral nucleus was performed. The results obtained from 15 p-endings presynaptic to 11 labeled tooth pulp afferent terminals were as follows. P-endings contained pleomorphic vesicles and made symmetrical synaptic contacts with labeled terminals. The p-endings showed small synaptic release-related ultrastructural parameters: volume, 0.82 ± 0.45 μm3 (mean ± SD); surface area, 4.50 ± 1.76 μm2; mitochondrial volume, 0.15 ± 0.07 μm3; total apposed surface area, 0.69 ± 0.24 μm2; active zone area, 0.10 ± 0.04 μm2; total vesicle number, 1045 ± 668.86; and vesicle density, 1677 ± 684/μm2. The volume of the p-endings showed strong positive correlation with the following parameters: surface area (r=0.97, P<0.01), mitochondrial volume (r=0.56, P<0.05), and total vesicle number (r=0.73, P<0.05). However, the volume of p-endings did not positively correlate or was very weakly correlated with the apposed surface area (r=-0.12, P=0.675) and active zone area (r=0.46, P=0.084). These results show that some synaptic release-related ultrastructural parameters of p-endings on the tooth pulp afferent terminals follow the “size principle” of Pierce and Mendell (1993) in the trigeminal nucleus oralis, but other parameters do not. Our findings may demonstrate a characteristic feature of synaptic release associated with p-endings.