Krox-20 is a C₂H₂-type zinc-finger transcription factor that plays an essential role in hindbrain development. The Krox-20 null mutation results in hindbrain anomalies that result in neonatal death due to respiratory and feeding deficits. Here we review our studies of how the Krox- 20 null mutation impacts the development of motor and sensory systems critical for the production of consummatory behaviors (suckling/chewing). First, we demonstrated that Krox-20 null mutants suffer a selective loss of primary jaw-opening muscles during prenatal development. In vivo and in vitro studies are reviewed that highlight intrinsic defects in mutant jaw-opener muscles that contribute to muscle degeneration. Next we focus on the impact of the mutation on proprioceptive neurons activated during consummatory behaviors. Mesencephalic trigeminal (Me5) neurons are primary sensory neurons that relay jaw proprioception to the central nervous system. These cells are unique because their cell bodies are located in the central as opposed to the peripheral nervous system. Data are reviewed that demonstrate the impact of the mutation on Me5 neurons, a cell group traditionally thought to emerge from the mesencephalon. We show that Krox-20 null mutants have twice as many Me5 neurons relative to wildtypes at E15, but by birth have half the number of Me5 cells as wildtypes. TUNEL assays performed in each set of studies reveal that Krox-20 expression acts to protect both muscle and mesencephalic trigeminal neurons against apoptosis, suggesting that Krox-20, in addition to its role in hindbrain patterning, has a broader, long-lasting role in development.

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Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca²⁺ phospholipids with high affinity and plays a role in Ca²⁺ regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expresse type II cell markers, PLCβ2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.

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Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (β-1, 3) N-acetylgalactosamine (galactosyl (β-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and α-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for α-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.

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Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel - which essentially contributes to the detection of pain sensation - in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.

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The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

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Neurotrophin-4/5 (NT-4/5) - a member of the neurotrophic factors - is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wild-type and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75- NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT- 4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.

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The present study examined whether mechanical stimulation is required for morphological maintenance of the Ruffini endings - primary mechanoreceptors in the periodontal ligament of the rat incisors, using a hypofunctional model by immunohistochemistry for protein gene product 9.5. The periodontal Ruffini endings of adult rats were observed to be restricted to the alveolar half of the lingual ligament where they displayed a dendritic arborization of expanded axon terminals with threadlike microprojections. In the experimental group, the tips of the upper and lower incisors were unilaterally ground to reduce mechanical stimulation of the ligament, i.e. occlusal force. A reduction in the occlusal force induced morphological changes in the terminal morphology of the periodontal Ruffini endings: they became smooth, unlike the irregular profiles exclusively observed in the control group. Quantitative analysis demonstrated significantly lower percentages of immunoreactive areas in the restricted portion on the ground sides than in normal animals. When incisor occlusion was re-established, the terminal portions of the Ruffini endings returned to their normal appearance, and the percentages of immunoreactive areas also recovered. The present results confirm the reduced size and number of axon terminals of periodontal Ruffini endings following reduced occlusal force and restoration of the morphological alteration after the re-establishment of incisor occlusion, indicating that proper mechanical stimulation is an important factor for maintaining the morphology of mechanoreceptors.

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The systemic administration of capsaicin is known to cause a massive loss of sensory primary neurons in newborn rats. Here we examined the trigeminal ganglion neurons immunohistochemically for the possible induction of activated forms of caspases-9 and -3 following a subcutaneous injection of capsaicin in newborn rats. The DNA fragmentation signal was labeled by a TUNEL method. TUNEL-positive neurons were rare (< 0.5%) at 24 h after injection of the vehicle without capsaicin. After the capsaicin injection, TUNEL-positive neurons began to increase by 12 h, reached a peak at 24 h (11.4%), and returned to the control level by 120 h. Vehicle control levels of caspase- 9-immunoreactive (ir) and caspase-3-ir neurons were low (< 0.5%). Neonatal capsaicin administration induced caspase-9-immunoreactivity (ir) and -3-ir. The temporal distributions of caspase-9-ir and caspase-3-ir neurons were similar to those of TUNEL-positive neurons with peak expressions at 24 h of 13.2 and 11.1%, respectively. A double-stain analysis at 24 h post-injection indicated 72% of TUNEL-positive neurons were caspase-9-ir, and 70% caspase-3-ir. Conversely, 78 and 68% of caspase-9-ir and caspase-3-ir neurons, respectively, were TUNEL-positive. Comparison of two adjacent sections immunostained for the two different antigens revealed the co-expression of the two caspases. These results suggest that neonatal capsaicin triggers the caspase cascade and, thereby, induces trigeminal primary neuronal apoptosis.

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The trigeminal subnucleus caudalis (Vc) is a critical relay site for processing nociceptive afferent input from the orofacial area in addition to its modulation by neuroplastic change. Although an administration of capsaicin in neonates induces a selective destruction of substance P (SP)-immunoreactive nerve fibers, little information is available regarding its detailed effects on the Vc, particularly during postnatal development. The present study examined postnatal changes in the distribution of SP in the Vc and trigeminal ganglion (TG) by immunohistochemical techniques in naïve (NV) and neonatally capsaicin-treated (CP) mice, combined with a quantitative analysis. The neonatal mice received a single subcutaneous injection of capsaicin (50 mg/kg) at 48 hours after birth. The neural density of the SP-immunoreaction decreased to approximately a quarter of that in 1-week-old NV mice but increased to three-quarters of that in the NV in the superficial area after postnatal week 2. A double staining with SP and myelin basic protein confirmed the absence of any SP-immunoreaction in the myelinated nerve fibers in both NV and CP mice. The SP-immunoreaction never overlapped with non-peptidergic IB4-labeled neurons in the Vc and TG of either group. Neither the size distribution of SP-positive neurons nor their relative ratio in the TG differed between NV and CP mice at the ages of postnatal weeks 1 and 8. These findings indicate two putative origins for the emergent SP-immunoreaction in the superficial layer of the Vc of the CP mice: the surviving trigeminal neurons with SP against capsaicin treatment and/or intrinsic neurons/interneurons in the Vc without SP under normal conditions.

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The prenatal development of neural circuits for rhythmical oral-motor behaviors used for feeding is essential for the survival of the newborn mammal. The N-methyl-D-aspartate (NMDA) receptor plays a critical role in brainstem circuits underlying postnatal oral-motor behaviors. To understand a role for the NMDA receptor in the emergence of sucking behavior we conducted physiological and immunohistochemical experiments using fetal rats. Physiology experiments examined the development of the NMDA dose response of the brainstem circuit responsible for generating rhythmical trigeminal activity by recording trigeminal motor outputs using an in vitro preparation. The high dose of NMDA agonist bath application affected the mean cycle duration of rhythmical trigeminal activity (RTA) at both embryonic day (E) 18-19 and E20-21 in comparison with standard concentration of NMDA agonist. NMDA receptor immunohistochemistry studies, using antibodies directed against subunits NR1, NR2A, NR2B, NR3A and NR3B were performed to determine the prenatal regulation of NMDA subunits in trigeminal motoneurons (Mo5), and mesencephalic trigeminal neurons (Me5) between E17 to E20. In Mo5, NR1, NR2A, NR2B and NR3A immunoreactivity was observed throughout the time frame sampled. NR3B immunoreactivity was not observed in Mo5 or Me5. In Mo5, there was a significant decrease in the percentage of NR2B immunoreactive neurons between E17 and E20, and a concurrent increase in the NR2A/NR2B ratio between E17 and E20. In Me5, NR1, NR2A and NR3A immunoreactivity was observed throughout the time frame sampled; a significant decrease in the percentage of NR2A immunoreactive neurons between E17 and E20, and NR3A immunoreactive neurons between E17 and E18 occurred. The timing of subunit changes between E17 and E18 is coincident with the prenatal emergence of rhythmical jaw movements, and in vitro rhythmical trigeminal activity, shown in earlier studies. Our data suggest that NMDA receptor plays an important role in the development and function of prenatal oral-motor circuits.

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