Selective distribution and function of primary afferent nociceptive inputs from deep muscle tissue to the brainstem trigeminal transition zone.

Orofacial injury activates two distinct regions in the spinal trigeminal complex, the subnuclei interpolaris/caudalis (Vi/Vc) transition zone and the laminated Vc, or medullary dorsal horn (MDH). Studies suggest that the Vi/Vc transition zone plays an important role in processing orofacial deep input. To test this hypothesis, we employed a double-tracing strategy to compare central projections of primary afferent neurons that innervate the masseter muscle and the overlying skin. Different tracers were injected either centrally (Fluoro-Gold: ventral Vi/Vc, or MDH) or peripherally (wheat germ agglutinin-conjugated horseradish peroxidase or cholera toxin B: masseter or overlying skin) in the same rat. Trigeminal ganglion tissue sections were processed for single or double immunohistochemistry. The double labeling of ganglion neurons indicates their site of peripheral and central innervations. A population of small to medium-sized neurons was doubly labeled after injections of the tracers into the masseter-Vi/Vc, masseter-MDH, or the skin-MDH. However, only a few double-labeled neurons were occasionally observed after injections of the tracers into the skin-Vi/Vc. Injection of an N-methyl-D-aspartate receptor antagonist, AP-5, into the Vi/Vc and MDH attenuated masseter inflammatory hyperalgesia. In contrast, hyperalgesia after inflammation of the skin overlying the masseter was attenuated by injection of AP-5 into the MDH but not Vi/Vc. These results indicate that while both masseter and cutaneous inputs project to the MDH, masseter afferents provide an additional input to the Vi/Vc. These findings provide further evidence to support a role of the trigeminal transition zone in response to orofacial deep injury.

Bilateral projection of functionally characterized trigeminal oralis neurons to trigeminal motoneurons in cats.

Intracellular Neurobiotin-injections were used to label functionally identified neurons in the rostro-dorsomedial part of the trigeminal oral nucleus (Vo.r) in the cat. The labeled Vo.r neurons with the mechanoreceptive field in oral tissues innervated bilaterally either jaw-opening motoneurons or jaw-closing motoneurons. This result suggests that Vo.r neurons play an important role in sensory-motor reflexes responsible for coordination of bilaterally symmetrical jaw movements.

Different physiological properties in a pool of mandibular closer motor neurons in a caterpillar, Bombyx mori.

To determine whether a large pool of mandibular closer motor neurons in an insect consists of different functional groups, the physiological properties of the motor neurons were investigated by intracellular recording techniques. The mandibular closer muscle of a caterpillar, Bombyx mori, was innervated by at least 11 motor neurons with large somata and 2-3 neurons with small somata. The large 11 motor neurons could be divided into slow and fast types on the basis of thresholds of neurons and frequency of soma spikes by current injections. The fast motor neurons usually evoked relatively larger muscle potentials, whereas the slow neurons evoked smaller potentials. The slow neurons spiked while the mandible was held in a fully closed position, whereas the fast neurons did not. The slow neurons did not spike while the mandible was free to move. These results suggest different physiological properties among closer motor neurons, supporting the hypothesis that the closer motor neurons in a large neuron pool can be divided into different functional groups.

Mesencephalic trigeminal sensory neurons of cat: axon pathways and structure of mechanoreceptive endings in periodontal ligament.

We injected 3H-proline into cat brainstem in order to label the entire mesencephalic trigeminal nucleus (Mes-V) for autoradiographic analysis of the size and pathways of Mes-V sensory axons and for microscopic study of Mes-V receptor structure in dental tissue. Labeled sensory axons were found in the trigeminal motor and sensory tracts and roots; approximately equal numbers of axons were found in both roots. The sensory root and all three divisions of the trigeminal nerve contained larger Mes-V axons than the motor root. Labeled Mes-V axons were found at the ganglion in the dorsomedial (infratrochlear) branch of the ophthalmic nerve but not in the ventrolateral branch. The mean diameter of Mes-V axons in periodontal ligament was 4.0 +/- 1.9 micron compared to 7.3 +/- 2.1 micron in maxillary and mandibular nerve, suggesting axonal arborization prior to innervation of ligament. Mes-V receptors in dental tissue were confined to ipsilateral periodontal ligament close to the root apex, with greater innervation on the posterior side. Receptor incidence was moderate for most teeth; however, maxillary first and second incisors and maxillary and mandibular canines had focal areas with remarkably dense innervation. No labeled axons were found in pulp of any ipsilateral teeth, and none was found in any contralateral dental tissue. EM-autoradiography demonstrated that Mes-V axons form unencapsulated Ruffini-like mechanoreceptors in periodontal ligament. The preterminal axons were small and myelinated. Neighboring bundles of unmyelinated axons and rare encapsulated endings were not labeled. The labeled mechanoreceptors branched to varying degrees among the ligament fibers; they contained numerous mitochondria and glycogen particles, as well as some vesicles and rare multivesicular bodies. They were surrounded by special Schwann cells that formed one or several layers around the ending. The endings were exposed to the basal lamina at numerous sites and occasionally extended fingers beyond the lamellar Schwann cells to contact ligament collagen.

Structural and functional aspects of the fast electrosensory pathway in the electrosensory lateral line lobe of the pulse fish Gymnotus carapo.

The fast electrosensory pathway (FEP) of gymnotiform fish is mediated by tuberous electroreceptor organs innervated by ganglion cells that synapse with spherical cells of the electrosensory lateral line lobe (ELL). Spherical cells project to the magnocellular mesencephalic nucleus. The electrosensory environment was represented somatotopically within ELL. The mandibular (MN) and the supraorbital (SON) nerves projected to rostral ELL (occupying 19-28% and 4-10%, respectively), and the posterior branch of the anterior lateral line nerve (pALLN) projected to caudal ELL (occupying 56-64%). Labeling with horseradish peroxidase or biotinylated dextran-amine demonstrated three kinds of synaptic endings coupling primary afferents to spherical cells: multiple synaptic knobs, medium-sized calyxes, and very large calyxes. Multiple synaptic knobs arose from MN and SON primary afferents and were found in a narrow rostral area covering the centrolateral (CLS) and lateral (LS) segments of ELL. Medium and large calyxes, proceeding from the same nerves, predominated in the remaining parts of the three segments of ELL containing spherical cells. Calyx-type endings were also found in the LS-occupying regions in which the pALLNs projected. Calyx-type endings formed gap junctions but also contained vesicles and showed submembrane specializations typical of chemical synapses. The postsynaptic spherical cells were linked by dendrosomatic gap junctions and were also contacted by unlabeled en passant synaptic boutons, whose fine structure suggested chemical transmission. Electrophysiological studies indicated that spherical cell responsiveness diminished after electrosensory stimulation. This apparently inhibitory phenomenon may be subserved by the unlabeled synaptic boutons, which possibly originate from interneurons that have yet to be identified.

Mandibular motor neurons of the caterpillar of the hawk moth Manduca sexta.

As part of a planned study of the central neural basis of feeding behaviour in larval Manduca sexta, the morphology and physiology of the mandibular motor system is here described. The gross neuroanatomy of the postoral head segments has been investigated, especially the course and structure of the mandibular nerves. The electrophysiology of the mandibular opener and closer muscles has been investigated by extra- and intracellular recording during feeding behaviour and during electrical stimulation of the motor nerve. All the muscle fibres examined are of the "fast," twitch type. Contraction is associated exclusively with locally or completely propagated overshooting action potentials, never with local junctional potentials. Control of the muscles is by recruitment of more motor units and/or an increase of frequency of action potentials. No inhibitory synaptic potentials could be found. The motor neurons of the mandibular muscles have been identified by cobalt backfills of the mandibular nerve, and characterized by intracellular recording and dye injection. There are 12 closer and 8 opener motor neurons. All motor neurons recorded so far evoke 1:1 twitches in the muscle, and none appear to be inhibitory. No GABA-immunoreactive axons could be found in the mandibular nerve.

Afferent activity from myelinated inferior alveolar nerve fibers in ferrets after constriction or section and regeneration.

To investigate possible peripheral mechanisms for post-injury sensory disorders in the trigeminal system, we have made electrophysiological recordings from myelinated fibres in the inferior alveolar nerve (IAN) which have previously sustained an injury. In earlier experiments we have shown that axons in ligature-induced neuromas of the IAN develop spontaneous activity and mechanical sensitivity. The present study has investigated these responses after two different types of injury. In 24 anaesthetised adult male ferrets the left IAN was either chronically constricted by four loose chromic gut ligatures (12 animals) or sectioned and regeneration permitted (12 animals). After recovery periods of 3 days, 1, 3, 6, 12 or 24 weeks, single unit recordings were made from the nerve proximal to the injury site. The proportion of units which were spontaneously active ranged from 0% to 19% after constriction injury and from 0% to 10% after nerve section and regeneration. Both groups revealed a marked variability between individual animals at similar time periods. Mechanical sensitivity was found in 0-42% of units after constriction and 0-25% of units after nerve section; both groups showed a significant negative correlation between mechanical sensitivity and recovery period. None of the fibres which had regained peripheral receptive fields was either spontaneously active or mechanically sensitive. There was no significant difference between the levels of spontaneous activity or mechanical sensitivity in the two groups or that previously found in ligature-induced neuromas. Thus we conclude that widely differing types of peripheral nerve injury are capable of initiating similar raised levels of afferent activity in myelinated inferior alveolar nerve fibres.

Sensory changes in the territory of the lingual and inferior alveolar nerves following lower third molar extraction.

Post-injury inflammation activates nociceptive systems and recruits normally non-nociceptive afferents into a pain processing role. During inflammation, Abeta low threshold mechanoreceptor afferents that usually mediate tactile sensation acquire properties of nociceptors, allowing them to participate in post-injury spontaneous pain and evoked abnormalities such as tenderness and pain to light touch. This study assessed the sensory consequences of post-injury inflammation following extraction of a single, lower third molar tooth. Extensive bilateral evaluations were performed in the territory of nerves assumed to be exposed to both inflammation and mechanical trauma, inflammation alone, or only the central consequences of peripheral inflammation. Testing at the distal termination of nerves assumed to be exposed to local inflammation (mental and lingual nerve territory) revealed decreased detection thresholds (P < 0.05) to electrical stimulation and to mechanical stimulation by sensitive, disposable filaments developed and validated for this application. Testing at sites of assumed inflammation and mechanical trauma (mental nerve territory) showed reduced pain thresholds to electrical stimulation. Thermal detection and pain thresholds were not altered at any location in patients, and no effects were observed in control subjects receiving only local anesthetic injections. These results in humans are consistent with recent experimental evidence that inflammatory processes alter the central consequence of activity in large-diameter Abeta touch primary afferents evoked under natural conditions by gentle mechanical stimulation. These effects result in hyperesthesia, increased sensitivity to light touch, and mechanical allodynia, pain evoked by normally innocuous stimulation of Abeta primary afferents.

Age-dependent changes in cat masseter nerve: an electrophysiological and morphological study.

The present study was undertaken to determine the manner in which aging affects the function and structure of the masseter nerve in old cats. Electrophysiological data demonstrated a significant decrease in the conduction velocity of the action potential in old cats compared with that observed in adult cats. Light microscopic analyses revealed an age-dependent decrease in axon diameter. Electron microscopic observations of the masseter nerve in the aged cats revealed a disruption of the myelin sheaths and a pronounced increase in collagen fibers in the endoneurium and perineurium. These morphological changes are discussed and then related to the decrease in conduction velocity which was observed in the electrophysiological portion of this study.

Effects of neonatal injury of the inferior alveolar nerve on the development and regeneration of periodontal nerve fibers in the rat incisor.

Our previous study showed that the migration of terminal Schwann cells occurred in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve (IAN) in the adult animals [Y. Atsumi, K. Matsumoto, M. Sakuda, T. Maeda, K. Kurisu, S. Wakisaka, Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: An immunohistochemical study on S-100 proteins, Brain Res. 849 (1999) 187-195]. The aim of the present study was to investigate the effects of neonatal transection of the IAN on the regeneration of axon elements and Schwann cells in the periodontal ligament of the rat lower incisor. Following transection of IAN at post-natal day 5 (PN 5d), when the numbers of both axon elements and the terminal Schwann cells were very small, regenerating nerve fibers appeared between post-injured days 7 (PO 7d) and PO 14d, and increased in number thereafter gradually. Although the terminal morphologies of regenerated Ruffini endings became identical to those of the adult animals by PO 54d, the number of regenerated PGP 9.5-IR nerve fibers did not recover the adult levels even by PO 56d. A small number of Schwann cells migrated into the shear zone, the border between the alveolus-related part (ARP) and the tooth-related part (TRP), but did not enter into the TRP. Following transection of the IAN at PN 14d or PN 28d, when clusters of apparent terminal Schwann cells could be recognized, axon regeneration started around PO 5d. Individual axon terminals of the regenerating Ruffini endings ramified and became identical to those of the adult animals around PO 28d, but the number of regenerated Ruffini endings was smaller than that of the adult animals. Similar to the adult animals, the migration of Schwann cells into the shear zone and TRP occurred, and disappeared prior to the completion of the axonal regeneration. The present results indicate that the migration of the Schwann cells into TRP during the regeneration of the periodontal nerve fibers following nerve injury to the IAN depends on the maturation of the terminal Schwann cells of the periodontal Ruffini endings, not on post-operative time.

Temporal analysis of neuropeptide Y expression in the rat trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve.

Temporal changes in the appearance and the cell-size spectrum of neuropeptide Y (NPY)-like immunoreactive (IR) cells in the rat trigeminal ganglion (TG) following peripheral axotomy of the inferior alveolar nerve (IAN) were studied by retrograde neuronal tracing with True Blue (TB) and immunohistochemistry for NPY. The number of labeled cells following application of TB to the cut-end of the IAN increased rapidly up to 3 days, and was maintained at a constant level thereafter. The size distribution of cross-sectional areas of TB-labeled cells was similar at 3 days and afterwards. NPY-IR cells, which were not detected in the normal TG, appeared on the first day following axotomy, and increased gradually in number reaching a maximum at 14 days. The frequency histogram of the cross-sectional areas of NPY-IR cells was similar at 3 days and afterwards. The present results indicate that the effect of nerve injury on the levels of NPY expression in the sensory neurons began soon after nerve injury, reaching a maximum around 14 days, and that induction of NPY in the sensory neurons occurred in the same cell size-specific manner for a long period.

Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection.

An increasing number of studies are suggesting that the adjacent uninjured primary afferents contribute to the mechanisms of neuropathic pain following peripheral nerve injury. In this study, we report that transection of the infraorbital nerve, a major branch of the maxillary nerve, causes exaggerated face grooming to normally innocuous mechanical stimuli in the skin territory of the uninjured mandibular nerve, and increases the expression of preprotachykinin mRNA in the primary afferent neurons in the mandibular zone in the trigeminal ganglia. Considering the various functions of substance P in the sensory transmission process, the increase in preprotachykinin mRNA in the uninjured primary afferent neurons may be one of the mechanisms of pain-related behavior in this neuropathic pain model.

Immunohistochemical analysis of neuronal nitric oxide synthase in the trigeminal ganglia of the crotaline snake Trimeresurus flavoviridis.

The expression of neuronal nitric oxide synthase (nNOS) in the trigeminal ganglia (TG) of the infrared-sensitive crotaline snake Trimeresurus flavoviridis was studied immunohistochemically. The percentage of nNOS-positive (+) neurons in the TG was significantly higher (about 3.5-fold, P<0.001) in the mandibular division than in the infrared-sensory processing area (the maxillary division and ophthalmic ganglion). nNOS was found in varying sizes of TG neurons. However, nNOS (+) neurons were more abundant in small and large neurons than in medium-sized neurons, which include most of the infrared-sensitive neurons of the TG. These findings suggest that nNOS may be involved in normal physiological functions, such as the transmission of tactile, vibrotactile, and nociceptive sensations in the TG, rather than in infrared sensory processing in this species.

Quantitative diameter analysis of lingual nerve axons in man.

Previous studies on axon counts and fiber-diameter spectra in lingual nerves have been carried out only on animal models. This study reports an histological investigation on a series of 20 lingual nerves removed post mortem from human subjects. The results show wide variation in the myelinated fiber counts--a variation which does not appear to be related to the ages of the subjects. When the results are compared with those of a previous study (Heasman and Beynon, 1983), it is seen that the lingual nerve:inferior dental nerve ratio of axon counts is not a consistent index. The fiber-diameter spectrum for the human lingual nerve is characterized by a bimodal curve with the more pronounced peak in the small-diameter fiber range.

Somatotopic organization and transmedian pathways of the rat trigeminal ganglion.

The localization of neuronal cell bodies in the trigeminal ganglion for the pulpal nerves of incisors and first and second molars in the rat was determined by utilization of retrograde axonal transport of horseradish peroxidase. Transmedian labeling was found with all teeth, but labeling was greater in the ipsilateral ganglion.

Myogenic determination and differentiation of the mouse palatal muscle in relation to the developing mandibular nerve.

The vertebrate palatal muscles are derived from the cranial paraxial mesoderm and start myogenesis by the expression of myogenic regulatory factors (MRFs). Predetermined myogenic cells migrate from the cranial paraxial mesoderm into the branchial arches, followed by myogenic differentiation. The objective of this study was to elucidate whether the determination, migration, and differentiation of myogenic cells during the myogenesis of the palatal muscles, particularly the tensor veli palatini (TVP), are related to the extending mandibular nerve in mouse embryos. By immunohistochemical staining at embryonic day (E) 9.5, MyoD1 and myogenin have been expressed in the mandibular arch, into which the mandibular nerve had not yet extended. At E11.5, these myogenic cells encircled the extending mandibular nerve and were distributed from the distal and lateral to the trigeminal ganglion and into the mandibular arch to form the muscle plate, a girdle-like structure. By E12.5, these myogenic cells lost their girdle-like pattern, vacated the trunk area of the mandibular nerve, and were separated into several incompletely divided masses encircling the collateral branches of the mandibular nerve. The TVP started differentiation at E13.5 with the appearance of myofilaments and acetylcholinesterase (AchE), whereas the other palatal muscles began differentiation at E14.5. We defined the differentiation process of mouse palatal muscles into five stages based on the present findings. These results suggest that the determination and initial migration of the palatal myogenic cells into the mandibular arch occur before the mandibular nerve extends out of the trigeminal ganglion, whereas the myogenic cells migrating into the final sites of differentiation intimately relate to the extending nerve.

Histometric study of myelinated fibers in the human trigeminal nerve.

The trigeminal ganglion, roots and the initial portion of the ophthalmic, maxillary and mandibular nerves were dissected in 3 cadavers, to study the number, area and composition of the fascicles, and the density and diameter spectra of myelinated fibers. The total number of fibers (x 1000) was 26 in the ophthalmic, 50 in the maxillary, and 78 in the mandibular division, 7.7 in the motor root and 170 in the sensory root. In all nerves, the histograms of fiber diameter had a bimodal distribution. Cutaneous and muscle nerve fascicles clearly differed in the fiber density and diameter. The ophthalmic and maxillary nerves (cutaneous) had similar fascicles, and their maximum fiber diameter averaged 14.5 microns. Most fascicles of the mandibular nerve (probably cutaneous fascicles) closely resembled those of the ophthalmic and maxillary nerves, but in some fascicles (probably muscle nerves) the fibers were larger, with a maximum diameter of 19.3 microns. The findings in the three peripheral divisions agree with electrophysiological data about sensory and motor conduction in human trigeminal nerves. The observation that the ophthalmic and maxillary nerves have similar fiber spectra indicates that a special fiber composition does not account for the sparing of the ophthalmic division in trigeminal neuralgia. The absence of very large (A alpha) fibers in the sensory root does not support the view that impulses from muscle spindles are conducted along this root.

Reconstructing the pathway of the tensor veli palatini motor nerve during early mouse development.

The motor axons innervating the tensor veli palatini (TVP) navigate a long distance from the trigeminal motor nucleus to their target. The pathway and time course of the TVP motor nerve during this navigation process remain poorly understood. The aim of this study was to elucidate the peripheral development of the TVP motor nerve, and to confirm when the morphological relationship is established between the nerve and target muscle progenitors. Using immunohistochemistry, carbocyanine fluorescent labeling, and computerized three-dimensional image-reconstruction methods, we demonstrated the development of the TVP motor nerve in mouse embryos. Further, the morphological relationship between the extending mandibular nerve and myogenic cells stained for MyoD1 was examined. The peripheral pathfinding of the TVP motor nerve was divided into three continuous stages: (1) the earliest trigeminal motor axons leave the metencephalon and enter the primordium of the trigeminal ganglion at E9.5, when MyoD1-positive cells can already be detected in the mesenchymal core of the mandibular arch; (2) converging with the sensory root, the trigeminal motor root excites the trigeminal ganglion and begins to approach the mandibular muscle precursors at E10.5; (3) collateral branching occurs at E12.5. By E13.5, a nerve branch splits from the mandibular nerve to innervate the TVP, which appears as an individual muscle mass. These results suggest that the early process of mandibular motor nerve extension is correlated with the trigeminal ganglion cells, whereas when growing out of the ganglion, the mandibular nerve has a close relationship with target myogenic cells throughout the later process of pathway finding.

Myelinated fibre diameters of human inferior alveolar nerves.

These were examined in a series of 36 human post-mortem specimens. One unimodal, 32 bimodal and 1 trimodal diameter curves were demonstrated; group analysis of all samples gave a bimodal curve with 2-4 and 8-9 microns peaks.

Response of Schwann cells in the inferior alveolar nerve to distraction osteogenesis: an ultrastructural and immunohistochemical study.

The biological mechanisms of nerve adaptation to distraction osteogenesis have not yet been elucidated. This study observed response of Schwann cells in the inferior alveolar nerve (IAN) following mandibular lengthening by electron microscopy and immunohistochemistry of S-100 protein, a specific marker of Schwann cells. Unilateral mandibular distraction (10mm elongation) was performed in nine young adult goats. Three animals were sacrificed at 7, 14 and 28 days after completion of distraction, respectively. The distracted IAN specimens and control nerves (from the contralateral sides) were harvested and processed for histological, ultrastructural and immunohistochemical examinations. Wallerian degeneration was observed in the distracted IAN, and Signs of axonal regeneration, as well as many activated Schwann cells were seen in the lengthened nerves. The expression of S-100 protein increased significantly at early stage of distraction osteogenesis, but almost returned to the normal level at 28 days after distraction. This study suggests that Wallerian degeneration caused by mechanical stretching may stimulate Schwann cells to enter a proliferated and activated state. Schwann cells and S-100 protein appear to play crucial roles in axonal regeneration that contributes to nerve adaptation to gradual distraction. Therefore, the IAN injury caused by mandibular gradual distraction was not serious; it seems to recover totally through a complicated repair mechanism.