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The trigeminal ganglion (TG) and mesencephalic trigeminal tract nucleus (Mes5) were investigated in wild type and dystonia musculorum (dt) mice to study the effect of dystonin deficiency on primary sensory neurons in the trigeminal nervous system. At postnatal day 14, the number of TG neurons was markedly decreased in dt mice when compared to wild type mice (43.1% reduction). In addition, dystonin disruption decreased the number of sensory neurons which bound to isolectin B4, and contained calcitonin gene-related peptide or high-affinity nerve growth factor receptor TrkA. Immunohistochemistry for caspase-3 demonstrated that dystonin deficiency induced excess cell death of TG neurons during the early postnatal period. In contrast, Mes5 neurons were barely affected in dt mice. These data together suggest that dystonin is necessary for survival of nociceptors but not proprioceptors in the trigeminal nervous system.

Immunohistochemistry for brain-derived neurotrophic factor (BDNF) was performed on the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 56.1+/-5.5%, 52.4+/-9.4% and 80.0+/-3.0% of sensory neurons, respectively, were immunoreactive for BDNF. These neurons were small- to medium-sized and observed throughout the ganglia. In the solitary tract nucleus, the neuropil showed BDNF immunoreactivity. A double immunofluorescence method demonstrated that BDNF-immunoreactive neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), P2X3 receptor, the capsaicin receptor (VR1) or vanilloid receptor 1-like receptor (VRL-1) in the jugular (CGRP, 43.5%; P2X3 receptor, 51.1%; VR1, 71.7%; VRL-1, 0.5%), petrosal (CGRP, 33.2%; P2X3 receptor, 58.4%; VR1, 54.2%; VRL-1, 23.3%) and nodose ganglia (CGRP, 1.8%; P2X3 receptor, 49.1%; VR1, 70.7%; VRL-1, 11.5%). The co-expression with tyrosine hydroxylase was also detected in the petrosal (2.9%) and nodose ganglia (2.2%). However, BDNF-immunoreactive neurons were devoid of parvalbumin in these ganglia. The present findings suggest that BDNF-containing vagal and glossopharyngeal sensory neurons have nociceptive and chemoreceptive functions.

The effect of neonatal masseteric nerve transection on primary proprioceptors was examined in the mesencephalic trigeminal tract nucleus (Mes5) of the rat. At 72 h to 21 days after the injury, the number of Mes5 neurons decreased on the side ipsilateral to the transection. The means+/-SD of percentage proportion of ipsilateral/contralateral neurons at 72 h and 21 days were 69.9+/-7.5% and 58.2+/-14.6%, respectively. The application of brain-derived neurotrophic factor to the proximal stump of the masseteric nerve delayed the loss of Mes5 neurons at 72 h after the injury; the mean numbers+/-SD of ipsilateral and contralateral Mes5 neurons in injured animals with BDNF application was 553.6+/-61.9 and 558.4+/-55.3, respectively. Saline application had no effect on the injury-induced loss of Mes5 neurons; i.e., the mean numbers+/-SD of ipsilateral and contralateral Mes5 neurons were 367.3+/-72.5 and 543+/-33.5, respectively. These findings indicate that trigeminal primary proprioceptors are sensitive to the neonatal injury. The survival of proprioceptors during early postnatal period is probably dependent upon brain-derived neurotrophic factor in the trigeminal nervous system.

The anterior part of the tongue was examined in wild type and dystonia musculorum mice to assess the effect of dystonin loss on fungiform papillae. In the mutant mouse, the density of fungiform papillae and their taste buds was severely decreased when compared to wild type littermates (papilla, 67% reduction; taste bud, 77% reduction). The mutation also reduced the size of these papillae (17% reduction) and taste buds (29% reduction). In addition, immunohistochemical analysis demonstrated that the dystonin mutation reduced the number of PGP 9.5 and calbindin D28k-containing nerve fibers in fungiform papillae. These data together suggest that dystonin is required for the innervation and development of fungiform papillae and taste buds.

Aspartate-immunoreactivity (ir) was examined in the mouse trigeminal ganglion (TG). The ir was detected in 34% of TG neurons and their cell bodies were of various sizes (mean +/- S.D. = 1,234 +/- 543 microm(2)). A triple immunofluorescence method revealed the co-expression of aspartate with calcitonin gene-related peptide (CGRP) and parvalbumin; 22% and 14% of aspartate-immunoreactive (ir) neurons were also immunoreactive for CGRP and parvalbumin, respectively. The co-expression of aspartate with both CGRP and parvalbumin was very rare in the TG. By retrograde tracing method, half and 66% of TG neurons which innervate the vibrissa and palate, respectively, contained aspartate-ir. The co-expression of aspartate with CGRP was more common among palatal neurons (36%) compared to vibrissal neurons (22%). Aspartate-ir neurons which co-expressed parvalbumin-ir were numerous in the vibrissa (17%) but not in the palate (4%). These findings may suggest that the function of aspartate-containing TG neurons is correlated with their peripheral receptive fields.

ASIC3-immunoreactivity (ir) was examined in the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 24.8%, 30.8% and 20.6% of sensory neurons, respectively, were immunoreactive for ASIC3. These neurons were observed throughout the ganglia. A double immunofluorescence method demonstrated that many ASIC3-immunoreactive (ir) neurons co-expressed calcitonin gene-related peptide (CGRP)- or vanilloid receptor subtype 1 (VRL-1)-ir in the jugular (CGRP, 77.8%; VRL-1, 28.0%) and petrosal ganglia (CGRP, 61.7%; VRL-1, 21.5%). In the nodose ganglion, however, such neurons were relatively rare (CGRP, 6.3%; VRL-1, 0.4%). ASIC3-ir neurons were mostly devoid of tyrosine hydroxylase in these ganglia. However, some ASIC3-ir neurons co-expressed calbindin D-28k in the petrosal (5.5%) and nodose ganglia (3.8%). These findings may suggest that ASIC3-containing neurons have a wide variety of sensory modalities in the vagal and glossopharyngeal sensory ganglia.

Immunohistochemistry for brain-derived neurotrophic factor (BDNF) was performed on the rat trigeminal ganglion (TG). The immunoreactivity (IR) was detected in 46% of TG neurons. These neurons were mostly small- or medium-sized (range, 149.7-1246.3 microm2; mean +/- SD = 373.4 +/- 151.6 microm2). A double immunofluorescence method also revealed that 54% of BDNF-immunoreactive (IR) neurons were immunoreactive for calcitonin-gene-related peptide. In addition, 93% of BDNF-IR TG neurons contained vanilloid receptor subtype 1. However, the co-expression of BDNF and vanilloid receptor 1-like receptor was very rare (less than 1%). In the trigeminal sensory nuclei, laminae II of the medullary dorsal horn was abundant in presumed BDNF-IR axon terminals. Such profiles were also detected in the dorsolateral part of the subnucleus oralis. The retrograde tracing and immunohistochemical methods demonstrated that BDNF-IR was common among cutaneous TG neurons (47%) but not tooth pulp TG neurons (13%). The present study indicates that BDNF-IR TG neurons have unmyelinated axons and project to the superficial medullary dorsal horn. It is likely that BDNF-containing neurons in both the trigeminal and spinal sensory systems have similarities in morphology and function. However, the content of BDNF in TG neurons probably depends on their peripheral targets. BDNF seems to convey nociceptive cutaneous input to the trigeminal sensory nuclei.

The distribution of gamma and beta subunits of epithelial Na(+) channel (ENaC), markers for low-threshold mechanoreceptors in peripheral tissues, was examined in the tooth pulp. In the root pulp, gammaENaC- and betaENaC-immunoreactive (IR) nerve fibers showed a thick smooth appearance. These nerve fibers ascended toward the pulp horn and formed subodontoblastic nerve plexuses. Immunoelectron microscopic method revealed that 63% of axons were immunoreactive for gammaENaC in the root pulp. Virtually all myelinated axons showed gammaENaC-IR (97%), whereas unmyelinated axons were mostly devoid of it (12%). These findings suggest that myelinated tooth pulp nociceptors respond to mechanical stimuli.

The co-expression of calretinin with parvalbumin and calbindin D-28k was examined in the rat cranial and spinal sensory ganglia by triple immunofluorescence method. In the trigeminal and nodose ganglia, 9% and 5% of calretinin-immunoreactive neurons, respectively, also contained both parvalbumin- and calbindin D-28k immunoreactivity. These neurons had large cell bodies. In the trigeminal ganglion, they were restricted to the caudal portion. Such neurons were evenly distributed throughout the nodose ganglion. The co-expression could not be detected in the dorsal root, jugular or petrosal ganglia. Nerve fibers which co-expressed all the three calcium-binding proteins were observed in the inferior alveolar nerve but not the infraorbital nerve or palate. In the periodontal ligament, these nerve fibers formed Ruffini-like endings. These findings suggest that (1) the co-expression in trigeminal neurons is intimately related to their peripheral receptive fields; (2) the three calcium-binding proteins (calretinin, parvalbumin, calbindin D-28k) co-expressed in the trigeminal neurons may have mechanoreceptive function in the periodontal ligament.

Capsaicin induces apoptosis in some types of neurons, but the exact molecular mechanism remains unclear. In this study, capsaicin was systemically administrated in newborn rats and the dorsal root ganglion (DRG) neurons were examined for caspase-immunoreactivity. Capsaicin-induced neuronal apoptosis was revealed by TUNEL. TUNEL-positive neurons rapidly increased, reaching the peak at 24 h post-injection when 10.6% of DRG neurons were apoptotic. Neurons expressing immunoreactivity for activated caspases-9 and -3 concomitantly increased. At 24 h, 15.9% and 17.7% of DRG neurons exhibited immunoreactivity for caspase-9 and caspase-3, respectively. DNA fragmentation signal and caspase-immunoreactivity were detected in less than 0.5% of DRG neurons of vehicle control rats. The immunoreactivity and TUNEL-positivity returned to the vehicle control level by 120 h. Double label immunohistochemistry revealed co-expression of caspase-9 and DNA fragmentation or caspase-3 and DNA fragmentation. These results suggest that the caspase cascade is involved in the primary neuronal apoptosis induced by neurotoxin capsaicin.

Immunohistochemistry for activated caspase-3 and terminal deoxynucleotidyl transferease-mediated dUTP-biotin nick end labeling (TUNEL) was performed on the trigeminal ganglion after infraorbital nerve transection in newborn rats. The injury induced caspase-3-immunoreactivity and DNA fragmentation in neuronal cell bodies in the maxillary division of the ganglion ipsilateral to the injury. Starting at 16 h post-injury the immunoreactive and TUNEL-positive neurons increased and reached the peak at 24 h (7.9% and 8.9%, respectively). Thereafter they decreased and returned to the normal control level (<<1%) by 72 h. A double staining procedure revealed coexpression of caspase-3-immunoreactivity and DNA fragmentation. 75.5% (114/151) of TUNEL-positive neurons expressed the immunoreactivity, while 84.4% (114/135) of immunoreactive neurons exhibited DNA fragmentation signal. These results suggest that caspase-3 plays an important role in apoptotic elimination of neonatally axotomized rodent primary neurons.

Parvalbumin- and calretinin-immunoreactivities (CR-irs) were examined in the molar tooth pulp of the rat using immunohistochemical methods. CR-ir fibers were further classified based on the tachykinin-ir revealed by a double immunofluorescence method. The rat root pulp contained three types of nerve fibers; parvalbumin-ir smooth fibers, CR-ir (TK-negative) smooth fibers and CR-ir (TK-ir) varicose fibers. These fibers projected toward the roof of the pulp chamber and pulp horn without marked ramification. In the subodontoblastic layer at the roof of the pulp chamber and pulp horn, parvalbumin-ir smooth fibers repeatedly ramified and extended varicose terminals into the odontoblastic layer. CR-ir (TK-negative) smooth fibers reached the subodontoblastic layer without marked ramification and gave rise to varicose terminals that appeared to terminate within the subodontoblastic layer. On the other hand, CR-ir (TK-ir) varicose fibers proceeded to the subodontoblastic layer at the roof of the pulp chamber and pulp horn, where they ramified and penetrated the odontoblastic layer. The present study indicates that the rat tooth pulp contains myelinated parvalbumin-ir and CR-ir (TK-negative) fibers, and unmyelinated CR-ir (TK-ir) fibers, and that they project varicose terminals to the subodontoblastic and odontoblastic layers. The central projection sites of these sensory fibers have yet to be revealed.

An investigation was made of the precise origin of the unmyelinated nerve fibers in the fungiform papillae of the bullfrog's tongue. Some unmyelinated nerve fibers in the fungiform papillae originate from the parasympathetic postganglionic cells in the glossopharyngeal nerve. Axonal enlargements of the parasympathetic nerve fibers were in close contact with the Merkel-like basal or supporting cells in the taste disk. These results seem to provide morphological evidence for the existence of an efferent control system in the taste disk.