R. Terayama

Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury.

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.

Brain-derived neurotrophic factor-immunoreactive primary sensory neurons in the rat trigeminal ganglion and trigeminal sensory nuclei.

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.

ASIC3-immunoreactive neurons in the rat vagal and glossopharyngeal sensory ganglia.

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.

Calretinin-containing neurons which co-express parvalbumin and calbindin D-28k in the rat spinal and cranial sensory ganglia; triple immunofluorescence study.

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.

Immunohistochemical localization of γ and β subunits of epithelial Na+ channel in the rat molar tooth pulp

Abstract The distribution of γ and β subunits of epithelial Na+ channel (ENaC), markers for low-threshold mechanoreceptors in peripheral tissues, was examined in the tooth pulp. In the root pulp, γENaC- and βENaC-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 γENaC in the root pulp. Virtually all myelinated axons showed γENaC-IR (97%), whereas unmyelinated axons were mostly devoid of it (12%). These findings suggest that myelinated tooth pulp nociceptors respond to mechanical stimuli.

Aspartate-immunoreactive primary sensory neurons in the mouse trigeminal ganglion.

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.

Dystonin deficiency reduces taste buds and fungiform papillae in the anterior part of the tongue

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.

The number of nociceptors in the trigeminal ganglion but not proprioceptors in the mesencephalic trigeminal tract nucleus is reduced in dystonin deficient dystonia musculorum mice

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.

PEPTIDE 19 IN THE RAT SUPERIOR CERVICAL GANGLION

Peptide 19 is a 7.6 kDa polypeptide which can bind to calmodulin and inhibit calcium-calmodulin signaling. In this study, peptide 19-immunoreactivity was examined in the rat superior cervical ganglion. In the ganglion, 54.8% of postganglionic sympathetic neuron profiles were immunoreactive for peptide 19. These neuron profiles were small- to medium-sized and measured 87-845 microm(2) (mean+/-SD = 343+/-111 microm(2)). Double immunofluorescence method revealed that 99.9% of peptide 19-containing neurons had neuropeptide Y in the superior cervical ganglion. Retrograde neuronal tracing and immunohistochemical studies also demonstrated that peptide 19 was common in postganglionic sympathetic neurons which innervated the facial skin and masseter but not the submandibular gland; 55.6% and 75.2% of cutaneous and muscular neuron profiles, respectively, contained peptide 19. Only 9.8% of glandular neurons were immunoreactive for peptide 19. These findings indicate that the content of peptide 19 in superior cervical ganglion neurons depends on their cell sizes and peripheral projections. On the other hand, colchicine injection into the superior cervical ganglion decreased the number of peptide 19-positive neurons (30.7%) compared to saline injection (53.3%). In contrast, the treatment induced nicotine adenine dinucleotide phosphate diaphorase activity in 12.7% of postganglionic sympathetic neurons. Double stain demonstrated that 56.3% of nicotine adenine dinucleotide phosphate diaphorase-positive neurons co-expressed peptide 19. These findings indicate that colchicine treatment causes decrease of peptide 19 expression and increase of nitric oxide synthase activity.

Activation of caspase cascade underlies the sensory primary neuronal apoptosis induced by neonatal capsaicin and peripheral nerve injury in rats

Basic fibroblast growth factor (bFGF) is commonly used to enrich and maintain neural stem cells in vitro; however, its precise role in neural stem cell renewal has remained elusive Olig2 is an essential transcription factor for oligodendrocyte lineage specification, and is predominantly expressed in ventral neuroepithelial cells in the medial and lateral ganglionic eminences (GE), where oligodendrocyte progenitors originate. We here demonstrate significant induction of Olig2 expression in dorsal neuroepithelium-derived cells cultured in the presence of bFGF. Among Olig2 expressing cells appearing after 5-day treatment with bFGF, 99.8% co-expressed nestin. There was no significant difference in proliferation or apoptosis in dorsal and ventral neuroepithelial cultures in the presence of bFGF, suggesting that bFGF induces ectopic expression of Olig2 in dorsal ‘cortical’ neuroepithelial cells. Similarly, expression of Mash1, another ventral neuroepithelial cell marker gene, was also induced in cultured dorsal neuroepithelial cells in the presence of bFGF. In contrast, the expression of dorsal neuroepithelial cell markers, such as Neurogenin1, Neurogenin2, Pax6 and Emx2, was down-regulated. These results suggested a possible ventralizing activity of bFGF, and in fact, bFGF-treated dorsal neuroepithelial cells acquired the potential to generate O4-positive oligodendrocytes with efficacy comparable to that observed with GE-derived cells. However, bFGF did not enable dorsal neuroepithelial cells to generate gamma-aminobutyric acid (GABA) neurons, which normally develop only from GE in vivo. Thus, bFGF endows dorsal telencephalic neural progenitors with ability to differentiate into oligodendrocytes but not GABAergic neurons, suggesting the presence of different mechanisms governing specification of dorsoventral cell identities of neuronal and glial cell lineages.