Nicotinamide adenine dinucleotide phosphate/neuronal nitric oxide synthase-positive neurons in the trigeminal subnucleus caudalis involved in tooth pulp nociception.

Sensory information on facial structures, including teeth pulp, periodontium, and gingiva, is relayed in the trigeminal complex. Tooth pulp inflammation constitutes a common clinical problem, and this peripheral injury can induce neuroplastic changes in trigeminal nociceptive neurons. There is considerable evidence that the trigeminal subnucleus caudalis (Vc) is the principal relay for trigeminal nociceptive information as well as modulation of the painful stimuli. Glutamatergic primary afferents innervating the tooth pulp project to the most superficial laminae of the Vc. N-methyl-D-aspartate receptor stimulation leads to the activation of the enzyme nitric oxide synthase (NOS), which synthesizes the free radical nitric oxide (NO). This enzyme is expressed mainly in lamina II interneurons, and in a small number of cells in lamina I as well as in deep laminae projection neurons of Vc. In the present study, we analyzed the temporal changes in neuronal NOS (nNOS) in Vc local circuitries after unilateral intermediate molar pulp injury. Our results demonstrate that a peripheral dental pulp injury leads to neuroplastic changes in the relative amount and activity of nNOS enzyme. Moreover, after a period of time, the nitrergic system shifts to the initial values, independently of the persistence of inflammation in the pulp tissues.

Involvement of nNOS in the antinociceptive activity of melatonin in inflammatory pain at the level of sensory neurons.

OBJECTIVE: The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain. MATERIALS AND METHODS: In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry. RESULTS: In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05). CONCLUSIONS: Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

Expression and colocalization of NADPH-diaphorase and heme oxygenase-2 in trigeminal ganglion and mesencephalic trigeminal nucleus of the rat.

Carbon monoxide (CO) and nitric oxide (NO) are two endogenously produced gases that can function as second messenger molecules in the nervous system. The enzyme systems responsible for CO and NO biosynthesis are heme oxygenase (HO) and nitric oxide synthase (NOS), respectively. The present study was undertaken to examine the distribution of HO-2 and NOS of the trigeminal primary afferent neurons of the rat, located in the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN), using histochemistry and immunohistochemistry. NADPH-d staining was found in most neurons in TG. The intensely NADPH-d-stained neurons were small- or medium-sized, while the large-sized neurons were less intensely stained. Immunocytochemistry for HO-2 revealed that almost all neurons in TG expressed HO-2, but they did not appear cell size-specific pattern. NADPH-d and HO-2 positive neurons appeared the same pattern, which was NADPH-d activity and HO-2 expression progressively declined from the caudal to rostral part of the MTN. A double staining revealed that the colocalization of NADPH-d/HO-2 neurons was 97.3% in TG and 97.6% in MTN. The remarkable parallels between NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and mediate the orofacial nociception and sensory feedback of the masticatory reflex arc together.

Synaptic Ras GTPase activating protein regulates pattern formation in the trigeminal system of mice.

The development of ordered connections or "maps" within the nervous system is a common feature of sensory systems and is crucial for their normal function. NMDA receptors are known to play a key role in the formation of these maps; however, the intracellular signaling pathways that mediate the effects of glutamate are poorly understood. Here, we demonstrate that SynGAP, a synaptic Ras GTPase activating protein, is essential for the anatomical development of whisker-related patterns in the developing somatosensory pathways in rodent forebrain. Mice lacking SynGAP show only partial segregation of barreloids in the thalamus, and thalamocortical axons segregate into rows but do not form whisker-related patches. In cortex, layer 4 cells do not aggregate to form barrels. In Syngap(+/-) animals, barreloids develop normally, and thalamocortical afferents segregate in layer 4, but cell segregation is retarded. SynGAP is not necessary for the development of whisker-related patterns in the brainstem. Immunoelectron microscopy for SynGAP from layer 4 revealed a postsynaptic localization with labeling in developing postsynaptic densities (PSDs). Biochemically, SynGAP associates with the PSD in a PSD-95-independent manner, and Psd-95(-/-) animals develop normal barrels. These data demonstrate an essential role for SynGAP signaling in the activity-dependent development of whisker-related maps selectively in forebrain structures indicating that the intracellular pathways by which NMDA receptor activation mediates map formation differ between brain regions and developmental stage.

Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat.

The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d(+)/CTb(+) or nNOS(+)/ CTb(+) double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 +/- 15 double-labeled neurons on the ipsilateral side and 33 +/- 14 on the contralateral side. CTb injections into the mXII resulted in 40 +/- 17 double-labeled neurons in this region on the ipsilateral side and 16 +/- 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 +/- 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons.

Increases in PKC gamma expression in trigeminal spinal nucleus is associated with orofacial thermal hyperalgesia in streptozotocin-induced diabetic mice.

Painful diabetic polyneuropathy (PDN) at the early phrase of diabetes frequently exhibits increased responsiveness to nociception. In diabetic patients and animal models, alterations in the transmission of orofacial sensory information have been demonstrated in trigeminal system. Herein, we examined the changes of protein kinase Cγ subunit (PKCγ) in trigeminal spinal nucleus (Sp5C) and observed the development of orofacial thermal sensitivity in streptozotocin (STZ)-induced type 1 diabetic mice. With hyperglycemia and body weight loss, STZ mice exhibited orofacial thermal hyperalgesia, along with increased PKCγ expression in Sp5C. Insulin treatment at the early stage of diabetes could alleviate the orofacial thermal hyperalgesia and impaired increased PKCγ in Sp5C in diabetic mice. In summary, our results demonstrate that PKCγ might be involved in orofacial thermal hyperalgesia of diabetes, and early insulin treatment might be effective way to treat orofacial PDN.

Effect of neonatal capsaicin and infraorbital nerve section on whisker-related patterns in the rat trigeminal nucleus.

In the present study, we investigated the effect of neonatally administered capsaicin on whisker-related pattern formation in the rat trigeminal complex. Both normal whisker-related patterns of barrelettes and the modified patterns seen after neonatal section of the infraorbital nerve were assessed. Capsaicin caused no change in the pattern or size of cytochrome oxidase (CO) barrelettes in the principal trigeminal nucleus (Vp) or trigeminal nucleus interpolaris (Vi) or caudalis (Vc). Injections of horseradish peroxidase (HRP) or wheatgerm agglutinin conjugated to HRP (WGA-HRP) into the posteroorbital (PO) whisker follicle in vehicle-treated animals showed that WGA labelled a larger number of trigeminal ganglion cells than HRP (203 +/- 23; cf. 158 +/- 19), with an increased labelling of small-diameter neurons (HRP: 25.9 +/- 7.7 microm; WGA: 23.2 +/- 7.2 pm). Capsaicin caused a loss of smaller diameter cells but had no effect on the location, cross-sectional area, or rostrocaudal extent of the transganglionically labelled HRP terminations in Vp, Vi, Vc, and cervical dorsal horn. WGA-HRP labelling revealed similar, but less dense, central terminal areas as HRP and an additional area of superficial terminals in the caudal medulla; these were also unaffected by capsaicin treatment. After infraorbital nerve section, CO patches and transganglionically labelled afferent terminations, corresponding to innervated nonmystacial whiskers, were approximately doubled in size. Capsaicin had no effect on the increased size of these spared whisker patches or their afferent terminal areas. These results suggest that barrelette formation is not dependent on unmyelinated afferents and that the changes in response properties seen after capsaicin, such as increased receptive fields, reflect functional changes rather than anatomical expansion of afferent terminal areas.

Development of the principal sensory nucleus of the trigeminal nerve of the rat and evidence for a transient synaptic field in the trigeminal sensory tract.

The early development of the principal sensory nucleus of the trigeminal nerve (PSN) was examined to determine whether spatiotemporal patterns of synaptogenesis coincide with patterns in neuronal generation, migration, and death. The morphogenesis of PSN neurons during the period from G16 to P14 was studied with a Golgi method. Prenatally, PSN neurons had dendrites that extended into the sensory tract of the trigeminal nerve (s5), and from as early as G18, these dendrites were studded with spines. The dendrites in the s5 degenerated or regressed in the early postnatal period so that the s5 was free of dendrites by P14. The development of anti-synapsin I immunoreactivity was traced from G14 to P10. Immunoreactive puncta (synaptic boutons) appeared in the medial third of the s5 transiently between G18 and P5. On the other hand, puncta in the PSN did not appear until G20, at which time they were confined to the lateral margin of the PSN. By P0, puncta were distributed throughout the PSN. Cytochrome oxidase activity in the PSN was low and unpatterned prenatally. Postnatally, cytochrome oxidase activity intensified and a segmented pattern of barreloids appeared in the ventral PSN on the day of birth. By P5, the complete pattern of barreloids, spanning the full width of the ventral PSN, was evident. The development of cytochrome oxidase activity in the PSN followed the lateral-to-medial gradient of synaptogenesis revealed by the development of synapsin 1 immunoreactivity. This gradient is opposite of that for neuronal generation, migration, and death. Moreover, the s5 serves as a transient synaptic field.

Regulation of neurotrophin-induced axonal responses via Rho GTPases.

Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons (Lentz et al. [1999] J. Neurosci. 19:1038-1048; Ulupinar et al. [2000a] J. Comp. Neurol 425:622-630). In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization (Ulupinar et al. [2000a] J. Comp. Neurol. 425:622-630). Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons.

On the innervation of trigeminal mesencephalic primary afferent neurons by adenosine deaminase-containing projections from the hypothalamus in the rat.

The localization and sources of adenosine deaminase-containing structures in the mesencephalic nucleus of the trigeminal nerve of the rat was studied using indirect immunofluorescence or immunoperoxidase immunohistochemical staining techniques for adenosine deaminase in combination with retrograde fluorescent tracing or lesion methods. The majority of large mesencephalic neurons were engulfed by a dense adenosine deaminase-immunoreactive plexus. Immunostaining was often punctate surrounding neuronal profiles or sometimes had the appearance of varicose fibers coursing over the neuronal surface. Occasionally, immunostained axons were found travelling towards and contacting mesencephalic neurons. Mesencephalic neuronal somas surrounded by immunofluorescence staining for adenosine deaminase were simultaneously labelled with fast blue after injections of this dye into the temporalis or masseter muscles of mastication. Injections of fast blue into the mesencephalic nucleus resulted in fast blue labelling of adenosine deaminase-immunoreactive neurons in the tuberal, caudal and postmammillary caudal magnocellular nuclei of the hypothalamus. Ablation of these hypothalamic nuclei caused a near total depletion of adenosine deaminase-immunostained fibers in the mesencephalic nucleus including those associated with mesencephalic neurons. It is concluded that adenosine deaminase-containing neurons in the posterior hypothalamus innervate mesencephalic primary sensory neurons, which are known to convey proprioceptive input to trigeminal motor nuclei controlling jaw muscles. The possibility is considered that the hypothalamus, via a direct action on these sensory neurons, may exert automatic control over jaw movements related to aggressive attack, defensive or feeding behavior. In addition, it appears that mesencephalic neurons may provide an ideal model system for electrophysiological investigations of the neurotransmitter(s) utilized by adenosine deaminase-containing hypothalamic projections.

Postnatal changes in cytochrome oxidase expressions in brain stem nuclei of rats: implications for sensitive periods.

Previously, we reported that cytochrome oxidase (CO) activity in the rat pre-Bötzinger complex (PBC) exhibited a plateau on postnatal days (P) 3-4 and a prominent decrease on P12 (Liu and Wong-Riley, J Appl Physiol 92: 923-934, 2002). These changes were correlated with a concomitant reduction in the expression of glutamate and N-methyl-d-aspartate receptor subunit 1 and an increase in GABA, GABAB, glycine receptor, and glutamate receptor 2. To determine whether changes were limited to the PBC, the present study aimed at examining the expression of CO in a number of brain stem nuclei, with or without known respiratory functions from P0 to P21 in rats: the ventrolateral subnucleus of the solitary tract nucleus, nucleus ambiguus, hypoglossal nucleus, nucleus raphe obscurus, dorsal motor nucleus of the vagus nerve, medial accessory olivary nucleus, spinal nucleus of the trigeminal nerve, and medial vestibular nucleus (MVe). Results indicated that, in all of the brain stem nuclei examined, CO activity exhibited a general increase with age from P0 to P21, with MVe having the slowest rise. Notably, in all of the nuclei examined except for MVe, there was a plateau or decrease at P3-P4 and a prominent rise-fall-rise pattern at P11-P13, similar to that observed in the PBC. In addition, there was a fall-rise-fall pattern at P15-P17 in these nuclei, instead of a plateau pattern in the PBC. Our data suggest that the two postnatal periods with reduced CO activity, P3-P4 and especially P12, may represent common sensitive periods for most of the brain stem nuclei with known or suspected respiratory control functions.

Early establishment of lesion-insensitive mature barrelettes corresponding to upper lip vibrissae in developing mice.

Vibrissae are tactile sense organs on the face of non-human mammals, and build up topographical representations in the brainstem trigeminal sensory nucleus called barrelettes. In the present study, we examined postnatal development of barrelettes corresponding to upper lip vibrissae by cytochrome oxidase (CO) histochemistry. At nuclear regions corresponding to upper lip vibrissae, a few segregated barrelettes first appeared at postnatal day 2 (P2), and segregation became clear for most upper lip barrelettes at P4. Compared with major barrelettes corresponding to mystacial vibrissae on the snout, the development of segregated pattern formation for upper lip barrelettes was retarded by 1-2 days. When vibrissa-related patterns were examined 5 days after infraorbital nerve transection, upper lip barrelettes became obscure in all mice lesioned at P1 and P2. Lesion-insensitive upper lip barrelettes first emerged in a few mice lesioned at P3 (33%), and the percentage attained 100% at P6. This temporal transition from lesion-sensitive to lesion-insensitive barrelettes was 3 days ahead of mystacial barrelettes. Therefore, upper lip barrelettes achieve rapid development within a narrow time frame during the first postnatal week. The early and rapid establishment of lesion-insensitive, mature barrelettes can be interpreted as suggesting the importance of oral sensory function in neonatal life.

Histochemical demonstration of vibrissae-representing patchy patterns of cytochrome oxidase activity within the trigeminal sensory nuclei in the cat.

Cytochrome oxidase histochemistry revealed patchy patterns of the enzyme activity in transverse sections through the caudal part of the ventral subnucleus of the principal sensory trigeminal nucleus, interpolar spinal trigeminal nucleus, and layer IV of the caudal spinal trigeminal nucleus in the cat. By the transganglionic transport method of horseradish peroxidase, the patterns were indicated to replicate the spatial array of the facial vibrissae.

Cytoarchitectonic correlates of the vibrissae in the medullary trigeminal complex of the mouse.

Cytoarchitectonic patterns in the medullary trigeminal complex of the mouse corresponding to mystacial vibrissae are described. These patterns are found within trigeminal sub-nuclei principalis, interpolaris and caudalis. The patterns are due to differential cell packing and are homeomorphic to the arrangement of the mystacial vibrissae on the face. The cytoarchitecture is similar, but complementary, to patterns of trigeminal afferents previously described using histochemical staining methods. Neonatal cautery of groups of vibrissae produces appropriate, specific and localized cytoarchitectural changes within all 3 trigeminal sub-nuclei.

The role of the principal sensory nucleus in central trigeminal pattern formation.

Complete lesions of the principal sensory nucleus in the neonatal rat disrupts vibrissae-related pattern formation in the ventral posterior nucleus of the dorsal thalamus. Similar lesions of the spinal trigeminal nucleus do not effect pattern formation in the ventral posterior nucleus. The results are interpreted as suggesting that the principal sensory nucleus provides a template for pattern formation in central trigeminal structures.

Immunohistochemical detection of heme oxygenase-2 in the periodontal Ruffini ending of the rat incisor.

The present study was carried out to examine the occurrence of heme oxygenase-2 (HO-2) in the periodontal ligament of the rat incisor. HO-2-like immunoreactive (-IR) structures showed dendritic profiles, resembling the Ruffini endings, in the alveolar half of the ligament of rat incisor. Neither thin nerve fibers nor perivascular nerve fibers displayed HO-2-like immunoreactivity (-LI). No non-neural elements exhibited HO-2-LI. Electron microscopy revealed that immunoreactions were diffusely observed in the axon terminals of the Ruffini endings, but neither terminal Schwann cells nor Schwann sheaths contained immunoreactions for HO-2. Both most neurons in the trigeminal ganglion and trigeminal mesencephalic nucleus showed HO-2-LI. The presence of HO-2 in the periodontal Ruffini endings and its absence in the periodontal thin nerve fibers suggest the involvement of carbon monoxide produced by HO-2 in mechanoreception in the periodontal ligament.

Current hypotheses of structural pattern formation in the somatosensory system and their potential relevance to humans.

Current hypotheses of structural pattern formation in the mammalian somatosensory system are modeled on experimental findings from the trigeminal system of rodents. The present results show that, like rodents, the trigeminal nucleus principalis of humans contains a parcellated pattern of cytochrome oxidase dense patches. These results provide an indication of the potential usefulness of rodent-based hypotheses for understanding pattern formation in human somatosensory connections.

Central terminals of orofacial primary afferents and NADPH-diaphorase activity in the trigemino-solitary complex of rats.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and the central terminal fields of branches of the mandibular and chorda tympani nerves were visualized histochemically at the same time using transganglionic transport of wheat germ agglutinin conjugated with horseradish peroxidase. The blue NADPH-d-positive neurons comprised a sparse network in the dorsomedial spinal trigeminal subnucleus oralis and a dense one in the rostral lateral division of the nucleus of the solitary tract. In the subnucleus caudalis, most labeled neurons were in the superficial zone, and smaller numbers were in the magnocellular zone. The NADPH-d-positive neurons in the subnucleus oralis and the nucleus of the solitary tract overlapped mostly with the transganglionically labeled terminal field from the lingual nerve, partly with the terminal field from the inferior alveolar and chorda tympani nerves, and rarely with the terminal field from the mental nerve. The NADPH-d-positive neurons in the dorsomedial paratrigeminal nucleus and subnucleus caudalis overlapped mostly with the terminal field from the lingual nerve, partly with the terminal field from the inferior alveolar and mental nerves and never with the terminal field from the chorda tympani. A statistically significant reduction in the number of NADPH-d-positive neurons was seen bilaterally in subnucleus oralis and the nucleus of the solitary tract when the lingual nerve was transected. Inflammatory insults to the lingual nerve or tooth pulps significantly increased the number of NADPH-d-positive neurons in subnucleus oralis, the nucleus of the solitary tract, and subnucleus caudalis. These results show that the NO/cyclic GMP system in the trigeminal and solitary nuclei is differentially regulated trans-synaptically by trigeminal afferents depending on the nucleus and sensory modality.

A specialized innervation of the tensor tympani muscle in Macaca fascicularis.

The innervation of the tensor tympani muscle of the middle ear in Macaca fascicularis (cynomolgus monkey) was studied using the horseradish peroxidase (HRP) neural tracing technique. A compact column of small trigeminal motoneurons was labeled ipsilaterally following intramuscular application of HRP to the tensor tympani muscle. This column is located ventral and lateral to the dorsolateral division of the trigeminal motor nucleus, and just medial to the descending trigeminal nerve rootlets. No labeled neurons were present in the trigeminal mesencephalic nucleus or any other brainstem nucleus. Results are compared with those previously reported in several non-primate mammalian species, and in detail with that of the cat. A possible differential role of the tensor tympani muscle in acoustic modulation/middle ear aeration between primate and non-primate mammals is discussed.

Distribution of beta-D-glucuronidase in the central nervous system of albino rats.

The distribution of beta-D-glucuronidase (GLR) in the central nervous system of albino rats was surveyed with a simultaneous-coupling histochemical technique. Specific neuronal populations displayed intense GLR activity. These groups include olfactory bulb mitral cells, neurones in the deep layer of the olfactory tubercle, cells in both the horizontal and vertical limbs of the nucleus of the diagonal band, paraventricular, supraoptic and suprachiasmatic nuclear neurons, cochlear and vestibular nuclear neurons, and facial, trigeminal and spinal motoneurons. Two types of intracellular GLR staining were observed. Granular staining was prominent in mitral cells; cytoplasmic staining and a perinuclear 'ring' were prominently stained in motoneurons. These patterns may correspond to lysosomal and endoplasmic reticular distribution of GLR in different cell types.