Yi Sul Cho

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disablility. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2−/−) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-d-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with d-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2−/− mice. Furthermore, treatment of Shank2−/− mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2−/− mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

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.

Quantitative analysis of afferents expressing substance P, calcitonin gene-related peptide, isolectin B4, neurofilament 200, and Peripherin in the sensory root of the rat trigeminal ganglion

Substance P (SP), calcitonin gene‐related peptide (CGRP), and isolectin B4 (IB4) are widely used as markers for peripheral neurons with unmyelinated fibers, whereas neurofilament 200 (NF200), and Peripherin are used as markers for neurons with myelinated fibers, and with unmyelinated or small‐caliber fibers, respectively. To study the selectivity of these markers for specific neuronal types, we analyzed their expression in neurons in the rat trigeminal ganglion by light‐ and electron‐microscopic immunocytochemistry. Most SP‐immunopositive (+), CGRP+, and IB4+ fibers were unmyelinated, but a small fraction (∼5%) were small myelinated fibers (<20 µm2 in cross‐sectional area, equivalent to <5 µm in diameter, Aδ fiber). Similarly, whereas the majority of NF200+ fibers were myelinated, a large fraction (23.9%) were unmyelinated, and whereas the majority of Peripherin+ fibers were unmyelinated and small myelinated, a significant fraction (15.5%) were large myelinated (>20 µm2 in cross‐sectional area, equivalent to >5 µm in diameter, Aβ fiber). Our findings confirm that SP, CGRP, and IB4 can be used as reliable markers for neurons with unmyelinated fibers, and question the suitability of NF200 as a marker for neurons with myelinated fibers, and of Peripherin as a marker for neurons with unmyelinated, or fine‐caliber fibers. J. Comp. Neurol. 523:126–138, 2015.

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.

An ultrastructural evidence for the expression of transient receptor potential ankyrin 1 (TRPA1) in astrocytes in the rat trigeminal caudal nucleus

The transient receptor potential ankyrin 1 (TRPA1) is implicated in the mechanical and cold hyperalgesia following inflammation and nerve injury. Its expression has been presumed to be confined to primary afferent terminals. Here, we show that TRPA1 is expressed in astrocytes in the superficial laminae of the rat trigeminal caudal nucleus by use of electron microscopic immunoperoxidase and immunogold labeling techniques. Immunoreactivity for TRPA1 was consistently observed in somata and process of astrocytes and was weaker than that in presumed nociceptive primary afferent terminals, but increased significantly in the fine process of astrocyte in rats with experimental inflammation of the temporomandibular joint. Thus, we provide ultrastructural evidence that TRPA1 is expressed in astrocytes in the brain stem and propose a novel pathway of its involvement in the central mechanism of inflammatory hyperalgesia.

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.

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.

Ultrastructural analysis of glutamate-, GABA-, and glycine-immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B–horseradish peroxidase (CTB‐HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate‐immunopositive boutons (69%) were more frequently observed than GABA‐ or glycine‐immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate‐immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA‐ or glycine‐immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate‐immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control.

Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

Background There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. Results The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. Conclusions These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.

Extrasynaptic homomeric glycine receptors in neurons of the rat trigeminal mesencephalic nucleus

The neurons in the trigeminal mesencephalic nucleus (Vmes) innervate jaw-closing muscle spindles and periodontal ligaments, and play a crucial role in the regulation of jaw movements. Recently, it was shown that many boutons that form synapses on them are immunopositive for glycine (Gly+), suggesting that these neurons receive glycinergic input. Information about the glycine receptors that mediate this input is needed to help understand the role of glycine in controlling Vmes neuron excitability. For this, we investigated the expression of glycine receptor subunit alpha 3 (GlyRα3) and gephyrin in neurons in Vmes and the trigeminal motor nucleus (Vmo), and the Gly+ boutons that contact them by light- and electron-microscopic immunocytochemistry and quantitative ultrastructural analysis. The somata of the Vmes neurons were immunostained for GlyRα3, but not gephyrin, indicating expression of homomeric GlyR. The immunostaining for GlyRα3 was localized away from the synapses in the Vmes neuron somata, in contrast to the Vmo neurons, where the staining for GlyRα3 and gephyrin were localized at the subsynaptic zones in somata and dendrites. Additionally, the ultrastructural determinants of synaptic strength, bouton volume, mitochondrial volume, and active zone area, were significantly smaller in Gly+ boutons on the Vmes neurons than in those on the Vmo neurons. These findings support the notion that the Vmes neurons receive glycinergic input via putative extrasynaptic homomeric glycine receptors, likely mediating a slow, tonic modulation of the Vmes neuron excitability.