Activation of transient receptor potential vanilloid 1 (TRPV1), a calcium permeable channel expressed in primary sensory neurons, induces the release of glutamate from their central and peripheral afferents during normal acute and pathological pain. However, little information is available regarding the glutamate release mechanism associated with TRPV1 activation in primary sensory neurons. To address this issue, we investigated the expression of vesicular glutamate transporter (VGLUT) in TRPV1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) under normal and complete Freund’s adjuvant (CFA)-induced inflammatory pain conditions using behavioral testing as well as double immunofluorescence staining with antisera against TRPV1 and VGLUT1 or VGLUT2. TRPV1 was primarily expressed in small and medium-sized TG neurons. TRPV1+ neurons constituted approximately 27% of all TG neurons. Among all TRPV1+ neurons, the proportion of TRPV1+ neurons coexpressing VGLUT1 (VGLUT1+/ TRPV1+ neurons) and VGLUT2 (VGLUT2+/TRPV1+ neurons) was 0.4% ± 0.2% and 22.4% ± 2.8%, respectively. The proportion of TRPV1+ and VGLUT2+ neurons was higher in the CFA group than in the control group (TRPV1+ neurons: 31.5% ± 2.5% vs. 26.5% ± 1.2%, VGLUT2+ neurons: 31.8% ± 1.1% vs. 24.6% ± 1.5%, p < 0.05), whereas the proportion of VGLUT1+, VGLUT1+/TRPV1+, and VGLUT2+/TRPV1+ neurons did not differ significantly between the CFA and control groups. These findings together suggest that VGLUT2, a major isoform of VGLUTs, is involved in TRPV1 activation-associated glutamate release during normal acute and inflammatory pain.

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.

Transient receptor potential melastatin 8 (TRPM8) plays a crucial role in innocuous cool sensation, acute cold pain and cold-induced hyperalgesia during pathologic conditions. To help understand TRPM8-mediated cold perception in the dental pulp and periodontal tissues, we examined the distribution of TRPM8-immunopositive (+) axons in molar and incisor pulp and periodontal tissues using transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. In the radicular pulp of the molar teeth, a small number of TRPM8+ axons were observed. TRPM8+ axons branched frequently and extensively in the core of coronal pulp, forming a network in the peripheral pulp. Some TRPM8+ axons ascended between odontoblasts and were observed in the dentinal tubule. TRPM8+ axons were linear-shaped in the radicular pulp, whereas many TRPM8+ axons showed portions shaped like beads connected with thin axonal stands at the peripheral pulp. TRPM8 was densely expressed in the bead portions. In the incisor pulp, TRPM8+ axons were occasionally observed in the core of the coronal pulp and rarely observed at the peripheral pulp. TRPM8+ axons were occasionally observed and showed a linear shape rather than a bead-like appearance in the periodontal ligament and lamina propria of the gingival tissue. These findings, showing differential distribution of TRPM8+ axons between radicular and coronal portions of the molar pulp, between incisor and molar pulp, and between dental pulp and periodontal tissues, may reflect differential cold sensitivity in these regions.

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.

The present study was aimed to evaluate the influence of glutaraldehyde (GA) concentration on multiple electron microscopic (EM) immunostaining using pre-embedding peroxidase and post-embedding immunogold method. Influence of various concentrations of GA included in the fixative on immuoreactivity was assessed in the multiple immunostaining using antisera against anti-transient receptor potential vanilloid 1 (TRPV1) for peroxidase staining and anti-GABA for immunogold labeling in the rat trigeminal caudal nucleus. Anti-TRPV1 antiserum had specificity in pre-embedding peroxidase staining when tissues were fixed with fixative containing paraformaldehyde (PFA) alone. Immunoreactivity for TRPV1 was specific in tissues fixed with fixative containing 0.5% GA at both perfusion and postfixation steps, though the immunoreactivity was weaker than in tissues fixed with fixative containing PFA alone. Tissues fixed with fixative containing 0.5% GA at the perfusion and postfixation steps showed specific immunogold staining for GABA. The results of the present study indicate that GA concentration is critical for immunoreactivity to antigens such as TRPV1 and GABA. This study also suggests that the appropriate GA concentration is 0.5% for multiple immunostaining with peroxidase labeling for TRPV1 and immunogold labeling for GABA.

Previous studies suggested that myelinated axons innervating rat molar pulps undergo morphological changes in their peripheral course. However, little information is available on the morphological feature of the parent axons at the site of origin. We therefore investigated the size of the myelinated parent axons and their morphological features at the proximal sensory root of the trigeminal ganglion by horseradish peroxidase (HRP) injection into rat upper molar pulps and subsequent light and electron microscopy. A total of 248 HRP-labeled myelinated axons investigated were highly variable in the size. Fiber area, fiber diameter, axon area (axoplasm area), axon diameter (axoplasm diameter), and myelin thickness were 11.32 ± 8.36 μm2 (0.80~53.17 μm2), 3.99 ± 1.53 μm (1.08~9.26 μm), 8.70 ± 6.30 μm2 (0.70~41.83 μm2), 3.13 ± 1.13 μm (0.94~7.20 μm) and 0.43 ± 0.23 μm (0.07~1.06 μm), respectively. The g-ratio (axon diameter / fiber diameter) of the labeled axons was 0.79 ± 0.05 (0.61~0.91). Axon diameter was highly correlated with myelin thickness (correlation coefficients, r=0.83) but little correlated with g-ratio (r=−0.33) of individual myelinated parent axons. These results indicate that myelin thickness of the myelinated parent axons innervating rat molar pulps increase with increasing axon diameter, thus maintaining a constant g-ratio.