Motohide Takemura

Difference in central projection of primary afferents innervating facial and intraoral structures in the rat

Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate was used to study the central projection of primary afferent neurons innervating facial and intraoral structures. The examined primary neurons innervating the facial structures were those comprising the frontal and zygomaticofacial nerves and those innervating the cornea, while the primary neurons innervating the intraoral structures included those innervating the mandibular incisor and molar tooth pulps and those comprising the palatine nerve. The primary afferents innervating the facial structures project to the lateral or ventral parts of the trigeminal principal, oral and interpolar subnuclei, and to the rostral cervical spinal dorsal horn across laminae I through V, with a greater proportion being directed to the spinal dorsal horn. The primary afferents innervating the intraoral structures terminate in the dorsomedial subdivisions of the trigeminal principal, oral and interpolar subnuclei, and in laminae I, II, and V of the medial medullary dorsal horn, with a much denser projection being distributed to the rostral subnuclei. In addition to the above brain stem trigeminal sensory nuclear complex, they project to the supratrigeminal nucleus, caudal solitary tract nucleus, and paratrigeminal nucleus. These observations agree with previously reported data that the central projection of trigeminal nerve is organized in different manners for the facial and intraoral structures. Furthermore, the present findings in conjunction with our previous studies clarify that the central projection of primary afferents from the facial skin is organized in a clear somatotopic fashion and that the terminal fields of primary afferents from the intraoral structures extensively overlap in the brain stem trigeminal nuclear complex particularly in its rostral subdivisions. The central mechanism of trigeminal nociception is discussed with particular respect to its difference between the facial and intraoral structures.

Topographic organization of central terminal region of different sensory branches of the rat mandibular nerve

The central projection of primary neurons comprising the auriculotemporal nerve, cutaneous branch of the mylohyoid nerve, inferior alveolar nerve, mental nerve, lingual nerve, and buccal nerve was investigated using transganglionic transport of HRP in young rats. In view of the topographic organization of central projection fields, the nerves were divided into two groups; i.e., those projecting to the dorsolateral margin of the trigeminal nucleus principalis, subnucleus oralis, and interpolaris (the auriculotemporal, mylohyoid, and mental nerves) and those projecting more medially (the inferior alveolar, lingual, and buccal nerves). The former group of nerves projected more caudally than the latter in the medullary and spinal dorsal horn complex rostral to the 3rd cervical segment, in general. Furthermore, the latter group projected to the nucleus of the solitary tract and the supratrigeminal and paratrigeminal nuclei, whereas the other nerves did not. The data indicate the following points: Primary neurons innervating the intraoral structures terminate medial (in trigeminal nucleus principalis and subnucleus oralis) and ventral (in subnucleus interpolaris) to the terminal fields of those innervating the facial skin. Primary neurons innervating the intraoral structures project to the nucleus of the solitary tract and the supra- and paratrigeminal nuclei, whereas those innervating the facial skin do not. Primary neurons innervating the periphery of the face project to the spinal dorsal horn and those innervating the intra/perioral region project to medullary dorsal horn, though this segregation from the medulla to the 3rd cervical segment is relatively loose. Only those trigeminal primary neurons, whose receptive fields extend to or beyond the midline, project to the contralateral dorsal horn from the medulla to the 3rd cervical segment.

BQ788, an endothelin ETB receptor antagonist, attenuates stab wound injury-induced reactive astrocytes in rat brain

Endothelins (ETs) are suggested to be involved in pathological or pathophysiological responses on brain injuries. In the present study, an involvement of ETs on activation of astrocytes in vivo was examined by using selective endothelin receptor antagonists. A stab wound injury on rat cerebral cortex increased immunoreactive ET‐1 at the injured site. GFAP‐positive [GFAP(+)] and vimentin‐positive [Vim(+)] cells appeared at the injured site in 1 day to 2 weeks after the injury. A continuous infusion of BQ788, a selective ETB receptor antagonist, into cerebral ventricle (23 nmole/day) attenuated increase in the numbers of GFAP(+) and Vim(+) cells after the injury. FR139317, a selective ETA antagonist (23 nmole/day), slightly decreased the number of Vim(+) cells but not that of GFAP(+) cells. Increase in the number of microglia/macrophages by a stab wound injury, which was determined by Griffonia simplicifolia isolectin B4 staining, was not affected by BQ788 and FR139317. These results suggest that activation of glial ETB receptors is one of the signal cascades leading to reactive astrocytes on brain injuries. GLIA 26:268–271, 1999.

ENDOTHELINS PROMOTE THE ACTIVATION OF ASTROCYTES IN RAT NEOSTRIATUM THROUGH ETB RECEPTORS

The effects of endothelin (ET)‐3 and an ETB receptor agonist on astrocytic activation in rat caudate putamen were examined by an immunohistochemical staining of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes. A single injection of 40 pmol ET‐3 into rat caudate putamen increased the number of GFAP positive cells compared to that in the contralateral saline‐injected side. Ala1,3,11,15‐ET‐1 (40 pmol), an ETB receptor agonist, also increased the number of striatal GFAP positive cells. The increases in GFAP positive cells were maximum (about 150% of the control side) in 1–2 weeks after injections of the ETs, and then reduced in 4 weeks. A continuous infusion of BQ788, an ETB receptor antagonist (23 nmol/day), into the lateral ventricle of the cerebrum antagonized the effect of Ala1,3,11,15‐ET‐1, while 80788 also reduced the number of GFAP positive cells in saline‐injected caudate putamen. Intrastriatal injection of 40 pmol Ala1,3,11,15‐ET‐1 did not affect the number of cells stained by B4 isolectin from Griffonia simplicifolia, which labels activated microglia/macrophages. Intraperitoneal administration of 5 mg/kg per day chloroquine and 0.2 mg/kg per day colchicine did not affect the action of Ala1,3,11,15‐ET‐1. These results suggest that activation of ETB receptors is involved in the induction of reactive astrocytes.

Cell size analysis of primary neurons innervating the cornea and tooth pulp of the rat

&NA; Primary neuronal cell bodies, whose peripheral axons comprised the cutaneous branch of the mylohyoid nerve or innervated the mandibular molar tooth pulp or the cornea, were labeled with HRP and their cross‐sectional area was analyzed. Most of their cell bodies were smaller than 1000 &mgr;m2 in cross‐sectional area and the histogram of each showed a unimodal pattern. The modes of percentage distribution were 100–200 &mgr;m2 (34.4%), 500–600 &mgr;m2 (17.4%) and 300–400 &mgr;m2 (35.1%) for the mylohyoid nerve, the tooth pulp and the cornea, respectively. A comparison of the 3 histograms indicated that there were at least 3 subpopulations of trigeminal primary neurons i.e., small, medium, and large cells. Electron microscopically, the large primary neurons innervating the tooth pulp had endoplasmic reticulum throughout the cytoplasm. The small primary neurons innervating the cornea showed a clear zonation of organelles and the endoplasmic reticulum was located in the periphery of the cytoplasm. The light microscopically identified small, medium and large cell groups may correspond to C‐, A&dgr;‐ and A&bgr;‐fibers. The tooth pulp and the cornea appear to receive mainly A&bgr;‐fibers and A&dgr;‐fibers, respectively. The cutaneous branch of the mylohyoid nerve appears to contain numerous C‐fibers and progressively smaller proportions of A&dgr;‐ and A&dgr;‐fibers.

Trigeminal transition zone/rostral ventromedial medulla connections and facilitation of orofacial hyperalgesia after masseter inflammation in rats

Recent studies have implicated a role for the trigeminal interpolaris/caudalis (Vi/Vc) transition zone in response to orofacial injury. Using combined neuronal tracing and Fos protein immunocytochemistry, we investigated functional connections between the Vi/Vc transition zone and rostral ventromedial medulla (RVM), a key structure in descending pain modulation. Rats were injected with a retrograde tracer, FluoroGold, into the RVM 7 days before injection of an inflammatory agent, complete Freunds adjuvant, into the masseter muscle and perfused at 2 hours postinflammation. A population of neurons in the ventral Vi/Vc overlapping with caudal ventrolateral medulla, and lamina V of the trigeminal subnucleus caudalis (Vc), exhibited FluoroGold/Fos double staining, suggesting the activation of the trigeminal‐RVM pathway after inflammation. No double‐labeled neurons were found in the dorsal Vi/Vc and laminae I–IV of Vc. Injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin, into the RVM resulted in labeling profiles overlapped with the region that showed FluoroGold/Fos double labeling, suggesting reciprocal connections between RVM and Vi/Vc. Lesions of Vc with a soma‐selective neurotoxin, ibotenic acid, significantly reduced inflammation‐induced Fos expression as well as the number of FluoroGold/Fos double‐labeled neurons in the ventral Vi/Vc (P < 0.05). Compared with control rats, lesions of the RVM (n = 6) or Vi/Vc (n = 6) with ibotenic acid led to the elimination or attenuation of masseter hyperalgesia/allodynia developed after masseter inflammation (P < 0.05–0.01). The present study demonstrates reciprocal connections between the ventral Vi/Vc transition zone and RVM. The Vi/Vc‐RVM pathway is activated after orofacial deep tissue injury and plays a critical role in facilitating orofacial hyperalgesia. J. Comp. Neurol. 493:510–523, 2005.

Tooth pulp primary neurons: Cell size analysis, central connection, and carbonic anhydrase activity

Cell bodies and central terminals of trigeminal primary afferent neurons innervating the mandibular molar and incisor tooth pulps were labeled by injecting various neuroanatomical tracers into these tooth pulps. The cell bodies of major constituents of primary neurons innervating the tooth pulp, cornea, and cutaneous branch of the mylohyoid nerve were large (> or = 500 microns2, 65%), medium (300-400 microns2, 35%), and small (100-200 microns2, 34%), respectively. Those innervating the tooth pulp (tooth pulp cells) had the Nissl pattern characteristic of large light cells of the A type, while those innervating the cornea were small dark cells of the B type. Thirty percent of the tooth pulp cells exhibited histochemically demonstrable carbonic anhydrase activity. The transganglionic transport of HRP-WGA indicated marked concentration of central terminals of the tooth pulp primaries in the rostral subdivisions of the brain stem sensory trigeminal nuclear complex. In contrast, central terminals of the corneal primaries were concentrated in the medullary dorsal horn.

Organization of the descending projections from the parabrachial nucleus to the trigeminal sensory nuclear complex and spinal dorsal horn in the rat

To clarify direct descending projections from the parabrachial nucleus (PB) to the trigeminal sensory nuclear complex (TSNC) and spinal dorsal horn (SpDH), the origin and termination of descending tract cells were examined by the anterograde and retrograde transport methods. Phaseolus vulgaris leucoagglutinin (PHA‐L) and Fluorogold (FG) or dextran‐tetramethylrhodamine (Rho) were used as neuronal tracers for the anterograde and retrograde transport, respectively. The ventrolateral PB, including Kölliker‐Fuse nucleus (KF), sent axons terminating mainly in the ventrolateral parts of rostral trigeminal nuclei of the principalis (Vp), oralis (Vo), and interpolaris (Vi) as well as in the inner lamina II of the medullary (nucleus caudalis, Vc) and SpDH. Although the descending projections were bilateral with an ipsilateral dominance, TSNC received a more dominant ipsilateral projection than SpDH. The cells of origin of the descending tracts were located mainly in KF, but TSNC received fewer projections from the KF than SpDH. Namely, TSNC received a considerable projection from the medial subnucleus of PB and the ventral parts of lateral subnuclei of PB, such as the central lateral subnucleus and lateral crescent area. The other difference noted between TSNC and SpDH was that the former received projections mainly from the caudal two thirds of KF and the latter from the rostral two thirds of KF. These results demonstrate the existence of direct parabrachial projections to TSNC and SpDH that are organized in a distinct manner and suggest that both pathways are involved in the control of nociception. J. Comp. Neurol. 383:94–111, 1997.

Electron microscopic observation of synaptic connections of jaw-muscle spindle and periodontal afferent terminals in the trigeminal motor and supratrigeminal nuclei in the cat

Previous studies indicate that the trigeminal motor nucleus (Vmo) and supratrigeminal nucleus (Vsup) receive direct projections from muscle spindle (MS) and periodontal ligament (PL) afferents. The aim of the present study is to examine the ultrastructural characteristics of the two kinds of afferent in both nuclei using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Our observations are based on complete or near‐complete reconstructions of 288 MS (six fibers) and 69 PL (eight fibers) afferent boutons in Vmo, and of 93 MS (four fibers) and 188 PL (four fibers) afferent boutons in Vsup. All the labeled boutons contained spherical synaptic vesicles and were presynaptic to neuronal elements, and some were postsynaptic to axon terminals containing pleomorphic, synaptic vesicles (P‐endings). In Vmo neuropil, MS afferent boutons were distributed widely from soma to distal dendrites, but PL afferent boutons predominated on distal dendrites. Most MS afferent boutons (87%) formed synaptic specialization(s) with one postsynaptic target while some (13%) contacting two or three dendritic profiles; PL afferents had a higher number of boutons (43%) contacting two or more dendritic profiles. A small but significant number of MS afferent boutons (12%) received contacts from P‐endings, but PL afferent boutons (36%) received three times as many contacts from P‐endings as MS afferents. In Vsup neuropil, most MS (72%) and PL (87%) afferent boutons formed two contacts presynaptic to one dendrite and postsynaptic to one P‐ending, and their participation in synaptic triads was much more frequent than in Vmo neuropil.

NADPH-diaphorase in the developing rat: Lower brainstem and cervical spinal cord, with special reference to the trigemino-solitary complex

A previous study indicated that in adult rat, a distinctive neuronal group in the dorsomedial division of the subnucleus oralis of the spinal trigeminal nucleus (SpVo) and the rostrolateral part of the nucleus of the solitary tract (Sn) is stained for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d), and suggested that the labeled structures are involved with sensorimotor reflexive functions. This study aimed to characterize the developmental expression of NADPH‐d in SpVo and Sn, including other areas of the lower brainstem and cervical spinal cord, by means of the enzyme histochemical staining technique, from the prenatal through the postnatal period. On embryonic day 12 (E12), no neurons in the brain were stained for NADPH‐d, whereas blood vessels were stained. Labeling in the vessels was consistently present throughout pre‐ and postnatal periods but decreased with development. On E15, labeled neurons appeared in the dorsomedial part of SpVo and the rostrolateral part of Sn, but not in the other nuclei. The labeled neurons in both nuclei increased in numbers drastically to E17. Postnatally, they tended to increase gradually in Sn, but to decrease slightly in SpVo. The cell size of labeled neurons reached a plateau at E17 in SpVo, but at postnatal day 4 (P4) in Sn. In other nuclei on E17, labeling appeared in the lateral paragigantocellular reticular, intermediate reticular, medullary reticular, pedunculopontine tegmental, and spinal vestibular nuclei, and laminae V, VI, and X of the cervical spinal cord. On E20 and P0, labeling appeared in the dorsal column, laterodorsal tegmental, raphe obscurus, parvocellular reticular, ventral gigantocellular reticular, and parahypoglossal nuclei, and laminae IX of the cervical spinal cord. On P4, labeling appeared in the parabrachial and median raphe nuclei, medial and caudolateral Sn, the magnocellular zone of subnucleus caudalis of the spinal trigeminal nucleus (SpVc), and laminae III/IV of the cervical spinal cord. On P10, labeling appeared in the paratrigeminal and dorsal raphe nuclei, the superficial zone of SpVc, and laminae I/II of the cervical spinal cord. No newly labeled neurons appeared in any nuclei after P14.