Sang Kyoo Paik

Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold and pungent compounds, is implicated in the mediation of nociception, but little is known about the processing of the TRPA1‐mediated nociceptive information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized the TRPA1‐positive (+) neurons in the trigeminal ganglion (TG) and investigated the distribution of TRPA1+ afferent fibers and their synaptic connectivity within the rat TSN and DH by using light and electron microscopic immunohistochemistry. In the TG, TRPA1 was expressed in unmyelinated and small myelinated axons and also occasionally in large myelinated axons. Many TRPA1+ neurons costained for the marker for peptidergic neurons substance P (26.8%) or the marker for nonpeptidergic neurons IB4 (44.5%). In the CNS, small numbers of axons and terminals were immunopositive for TRPA1 throughout the rostral TSN, in contrast to the dense network of positive fibers and terminals in the superficial laminae of the trigeminal caudal nucleus (Vc) and DH. The TRPA1+ terminals contained clear round vesicles, were presynaptic to one or two dendrites, and rarely participated in axoaxonic contacts, suggesting involvement in relatively simple synaptic circuitry with a small degree of synaptic divergence and little presynaptic modulation. Immunoreactivity for TRPA1 was also occasionally observed in postsynaptic dendrites. These results suggest that TRPA1‐dependent orofacial and spinal nociceptive input is processed mainly in the superficial laminae of the Vc and DH in a specific manner and may be processed differently between the rostral TSN and Vc. J. Comp. Neurol. 518:687–698, 2010.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat

Trigeminal primary afferents expressing P2X3 receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X3‐immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X3 immunoreactivity was mainly in small and medium‐sized somata, but also frequently in large somata. Although most P2X3+ somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X3+ fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X3 immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X3+ terminals contained numerous clear, round vesicles and sparse large, dense‐core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X3+ terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X3+ afferents to all TBSN and that it may be processed differently in different TBSN. J. Comp. Neurol. 506:627–639, 2008.

Light and electron microscopic analysis of the somata and parent axons innervating the rat upper molar and lower incisor pulp.

The morphology of intradental nerve fibers of permanent teeth and of continuously growing rodent incisors has been studied in detail but little information is available on the parent axons that give rise to these fibers. Here we examined the axons and somata of trigeminal neurons that innervate the rat upper molar and lower incisor pulp using tracing with horseradish peroxidase and light and electron microscopic analysis. The majority (approximately 80%) of the parent axons in the proximal root of the trigeminal ganglion that innervated either molar or incisor pulp were small myelinated fibers (<20 microm(2) cross-sectional area). The remaining approximately 20% of the fibers were almost exclusively large myelinated for the molar pulp and unmyelinated for the incisor pulp. The majority of neuronal somata in the trigeminal ganglion that innervated either molar (48%) or incisor pulp (62%) were medium in size (300-600 microm(2) cross-sectional area). Large somata (>600 microm(2)) constituted 34% and 20% of the trigeminal neurons innervating molar and incisor pulp, respectively, while small somata (<300 microm(2)) constituted 17% of the molar and 18% of the incisor neurons. The present study revealed that the morphology of parent axons of dental primary sensory neurons may differ from that of their intradental branches, and also suggests that the nerve fiber function may be carried out differently in the molar and incisor pulp in the rat.

Expression of Metabotropic Glutamate Receptor mGluR5 in Human Dental Pulp

Accumulating evidence indicates that the metabotropic glutamate receptor mGluR5 is involved in the peripheral mechanisms of inflammatory nociception. To investigate whether mGluR5 may mediate the inflammatory pain and thermal hyperalgesia in the dental pulp, we examined the expression of mGluR5 and transient receptor potential vanilloid 1 (TRPV1) in human dental pulp by immunohistochemistry and electron microscopy; mGluR5-immunopositive (+) axons were observed in nerve bundles and branched extensively within the peripheral coronal pulp. Most of the mGluR5+ axons were unmyelinated. A large fraction of these axons (36.5%) were immunostained for TRPV1. Immunoreactivity for mGluR5 and TRPV1 was also observed in odontoblasts. These results support the possibility that the nerve fibers in the dental pulp mediate inflammatory pain and thermal hyperalgesia through coactivation of mGluR5 and TRPV1 and also suggest a possible role for odontoblasts in the transduction of nociceptive signals via mGluR5-mediated mechanism.

The distribution of inhibitory and excitatory synapses on single, reconstructed jaw-opening motoneurons in the cat

In a previous study, we reported that the distribution of inhibitory input, in contrast to excitatory input, decreased somatofugally along dendrites of cat jaw-closing alpha-motoneurons [J Comp Neurol 414 (1999) 454]. The present study examined the distribution of GABA, glycine, and glutamate immunopositive boutons covering horseradish peroxidase-labeled cat jaw-opening motoneurons. The motoneurons were divided into four compartments: the soma, and primary, intermediate, and distal dendrites. Ninety-seven percent of the total number of studied boutons had immunoreactivity for at least one of the three amino acids. The proportion of boutons immunoreactive for GABA and/or glycine was lower than the proportion of boutons immunoreactive for glutamate. Boutons immunoreactive to glycine alone were more numerous than boutons double-labeled for GABA and glycine, which, in turn, occurred more frequently than boutons immunoreactive to GABA alone. The percentage synaptic covering (proportion of membrane covered by synaptic boutons) of the putatively excitatory (glutamate containing) and putatively inhibitory (GABA and/or glycine containing) boutons decreased somatofugally along the dendrites. Such systematic variations were not seen in the packing density (number of boutons per 100 microm(2)); the packing density showed a distinct drop between the soma and primary dendrites but did not differ significantly among the three dendritic compartments. Overall, the packing density was slightly higher for the putatively excitatory boutons than for the inhibitory ones. When taken together with previous analyses of jaw-closing alpha-motoneurons the present data on jaw-opening alpha-motoneurons indicate that the two types of neuron differ in regard to the nature of synaptic integration in the dendritic tree.

GABA and glycine in synaptic microcircuits associated with physiologically characterized primary afferents of cat trigeminal principal nucleus

Previous studies suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by GABA-mediated presynaptic mechanisms in the trigeminal principal sensory nucleus (Vp) than other sensory nuclei. However, it is unknown if such a mechanism is common to functionally different classes of primary afferent in the same nucleus or across the nuclei. To address these issues, the present study focused on synaptic microcircuits associated with slowly adapting (SA) mechanosensory afferents innervating the periodontal ligaments in the cat Vp and attempted to examine GABA, glycine, and glutamate immunoreactivity in axon terminals involved in the circuits. Afferents were physiologically characterized before injection of horseradish peroxidase (HRP) and preparation for electron microscopy. HRP-labeled afferent boutons were serially sectioned and immunostained with antibodies against GABA, glycine, and glutamate using a postembedding immunogold method. All the afferent boutons examined contacted non-primary dendrites and they were frequently postsynaptic to unlabeled axons (p-endings). Axodendritic and axoaxonic contacts per afferent bouton were 1.3 (46/35) and 2.0 (70/35), respectively. Most p-endings were immunoreactive for GABA (63/70) and also glycine was co-stained in the majority of the p-endings (49/63). Thirty percent of p-endings with the colocalization of GABA and glycine participated in synaptic triads where a p-ending formed a synapse with the same dendrite as the afferent bouton. None of the p-endings was immunoreactive for glutamate. Most afferent boutons were enriched with glutamate but were immunonegative for GABA and glycine. This study provides evidence suggesting that transmission from SA afferents is strongly controlled presynaptically by GABAergic interneurons with colocalized glycine, and that a proportion of these interneurons, involved in synaptic triads, may also have postsynaptic inhibitory actions on target neurons of the SA afferents.

The synaptic microcircuitry associated with primary afferent terminals in the interpolaris and caudalis of trigeminal sensory nuclear complex.

Previous ultrastructural studies indicating a higher number of axoaxonic contacts on individual low-threshold mechanoreceptive afferents in the principalis (Vp) than in the oralis (Vo) of cat trigeminal sensory nuclear complex (TSNC) suggest that the synaptic microcircuitry associated with primary afferents manifests unique differences across the sensory nuclei of TSNC. To address this issue, we analyzed synaptic microcircuits associated with fast adapting vibrissa afferent terminals in the interpolaris (Vi) and caudalis (Vc, laminae III/IV) by using intraaxonal injections of horseradish peroxidase (HRP) in cats. Forty-two and 65 HRP-labeled boutons were analyzed in the Vi and Vc, respectively. The labeled boutons contained clear, spherical vesicles. They most frequently formed asymmetric axodendritic synapses and were commonly postsynaptic to unlabeled axon terminals containing pleomorphic vesicles (p-endings) with symmetric junctions. The examination of synaptic contacts over the entire surface of individual boutons indicated that the afferent boutons made contacts with an average of two postsynaptic targets in the Vi and Vc. In contrast, axoaxonic contacts, and labeled boutons participating in synaptic triads, where p-endings contacted both the boutons and their postsynaptic targets, were, on average, higher in the Vi than in the Vc. These results suggest that the output of sensory information conveyed through low-threshold mechanoreceptive afferents is more strongly controlled at the level of the first synapse by presynaptic and postsynaptic mechanisms in the Vi responsible for sensory discriminative functions than in the Vc for sensorimotor reflexive functions.

Ultrastructural analysis of the synaptic connectivity of TRPV1‐expressing primary afferent terminals in the rat trigeminal caudal nucleus

Trigeminal primary afferents that express the transient receptor potential vanilloid 1 (TRPV1) are important for the transmission of orofacial nociception. However, little is known about how the TRPV1‐mediated nociceptive information is processed at the first relay nucleus in the central nervous system (CNS). To address this issue, we studied the synaptic connectivity of TRPV1‐positive (+) terminals in the rat trigeminal caudal nucleus (Vc) by using electron microscopic immunohistochemistry and analysis of serial thin sections. Whereas the large majority of TRPV1+ terminals made synaptic contacts of an asymmetric type with one or two postsynaptic dendrites, a considerable fraction also participated in complex glomerular synaptic arrangements. A few TRPV1+ terminals received axoaxonic contacts from synaptic endings that contained pleomorphic synaptic vesicles and were immunolabeled for glutamic acid decarboxylase, the synthesizing enzyme for the inhibitory neurotransmitter γ‐aminobutyric acid (GABA). We classified the TRPV1+ terminals into an S‐type, containing less than five dense‐core vesicles (DCVs), and a DCV‐type, containing five or more DCVs. The number of postsynaptic dendrites was similar between the two types of terminals; however, whereas axoaxonic contacts were frequent on the S‐type, the DCV‐type did not receive axoaxonic contacts. In the sensory root of the trigeminal ganglion, TRPV1+ axons were mostly unmyelinated, and a small fraction was small myelinated. These results suggest that the TRPV1‐mediated nociceptive information from the orofacial region is processed in a specific manner by two distinct types of synaptic arrangements in the Vc, and that the central input of a few TRPV1+ afferents is presynaptically modulated via a GABA‐mediated mechanism. J. Comp. Neurol. 518:4134–4146, 2010.

Developmental changes in distribution of γ-aminobutyric acid- and glycine-immunoreactive boutons on rat trigeminal motoneurons. I. Jaw-closing motoneurons

We have previously described the distribution pattern of inhibitory synapses on rat jaw‐closing (JC) α‐ and γ‐motoneurons. In the present study, we investigated developmental changes in inhibitory synapses on JC motoneurons. We performed a quantitative ultrastructural analysis of putative inhibitory synaptic boutons on JC motoneuron somata by using postembedding immunogold labeling for GABA and glycine. In total, 206, 350, and 497 boutons contacting JC motoneuron somata were analyzed at postnatal days 2 (P2), 11 (P11) and 31 (P31), respectively. The size of the somata increased significantly during postnatal development. The size distribution was bimodal at P31. Mean length of the boutons and percentage of synaptic covering also increased during postnatal development, whereas bouton density did not differ significantly among the three age groups. Synaptic boutons on the somata of JC α‐motoneurons could be classified into four types: boutons immunoreactive for 1) GABA only, 2) glycine only, 3) both GABA and glycine, and 4) neither GABA nor glycine. There was no developmental change in the proportion of putative inhibitory boutons to the total number of studied boutons. However, the glycine‐only boutons increased significantly (15.1% to 27.3%), and the GABA‐only boutons decreased significantly (17.7% to 2.6%) during the period from P11 to P31. Our ultrastructural data indicate that the inhibitory synaptic input to JC motoneurons is developmentally regulated and that there is a postnatal switch from GABA to glycine. The postnatal changes revealed in the present study could play an important role in the maturation of the oral motor system. J. Comp. Neurol. 503:779–789, 2007.

Quantitative ultrastructural analysis of the neurofilament 200-positive axons in the rat dental pulp.

INTRODUCTION Previous studies have suggested that myelinated axons lose their myelin and become thinner in their peripheral course to the target organ. In this study, we investigated the morphologic changes of pulpal myelinated axons between their root portion (radicular pulp) and their terminal area (peripheral pulp). METHODS Sections of pulp of the rat upper molar teeth were immunostained for the marker of myelinated axons neurofilament (NF) 200. The proportion of NF200+ myelinated and unmyelinated fibers and their sizes were analyzed by using quantitative electron microscopy. RESULTS The axon area, myelin thickness, and fraction of NF200+ myelinated axons of all NF200+ axons were significantly lower in peripheral than in radicular pulp. In addition, large unmyelinated axons were frequently observed in peripheral pulp. CONCLUSIONS These results suggest that pulpal innervation originates predominantly from myelinated axons, and the myelinated axons undergo extensive morphologic changes during their course from the radicular to the peripheral pulp.