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Tropomyosin receptor kinase A (trkA), a high affinity receptor for nerve growth factor (NGF), has been implicated in neuronal survival, neurite outgrowth and inflammatory pain. So far, the characterization of the primary sensory neurons that express trkA, and are thus potentially affected by NGF, has remained incomplete. The goal of this study was to investigate the trkA-expressing neurons and fibers in the rat trigeminal ganglion and its sensory root using light- and electron-microscopic immunohistochemistry and quantitative analysis. TrkA-immunopositive (+) trigeminal neurons varied from small to large. Double immunofluorescent staining showed that about 28%, 33% and 3% of the trkA(+) neurons coexpressed SP, CGRP and IB4, respectively. About 11% of the trkA(+) neurons also coexpressed parvalbumin. Electron microscopy revealed that trkA was expressed in all types of fibers: While the large majority of the trkA(+) fibers were unmyelinated (35.3%) and small myelinated (<20 μm 2 in cross-sectional area; 45.5%), a still considerable fraction (19.2%) was large myelinated. These findings indicate that all types of trigeminal neurons (ones with unmyelinated, small myelinated or large myelinated fibers) may be regulated by NGF/trkA signaling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Parvalbumin (PV), calretinin (CR), calbindin D-28k (CB), stage specific embryonic antigen-4 (SSEA4), and phosphorylated neurofilament 200 (pNF200) have been commonly used as markers for primary afferent neurons with large myelinated (A) fibers but detailed information on the expression of these markers in specific primary afferent fiber types is still lacking. We here examined the fibers that express PV, CR, CB, SSEA4, and pNF200 in the trigeminal ganglion and its peripheral sensory root by light- and electron-microscopic immunohistochemistry and quantitative analysis. We found that all CR-immunopositive (+), CB+, and SSEA4+ fibers and virtually all (98.8%) PV+ fibers were myelinated, most CR+ fibers were large myelinated, whereas most CB+ and SSEA4+ fibers were small myelinated. One half of the PV+ fibers were small myelinated and the other half were large myelinated. Of all pNF200+ fibers, about a third each were small myelinated, large myelinated, and unmyelinated. These findings suggest that PV, CR, CB, and SSEA4 can be used as specific markers for primary afferent neurons with myelinated fibers, but that pNF200 is not suitable as a specific marker for primary afferent neurons with myelinated fibers, and also raise the possibility that PV, CR, CB, and SSEA4 may be expressed in both mechanoreceptive and nociceptive neurons., (© 2018 Wiley Periodicals, Inc.)

Stromal cells/telocytes (SCs/TCs) were recently described in the human adult trigeminal ganglion (TG). As some markers are equally expressed in SCs/TCs and endothelial cells, we hypothesized that a subset of the TG SCs/TCs is in fact represented by endothelial progenitor cells of a myelomonocytic origin. This study aimed to evaluate whether the interstitial cells of the human adult TG correlate with the myelomonocytic lineage. We used primary antibodies for c-erbB2/HER-2, CD31, nestin, CD10, CD117/c-kit, von Willebrand factor (vWF), CD34, Stro-1, CD146, α-smooth muscle actin (α-SMA), CD68, VEGFR-2 and cytokeratin 7 (CK7). The TG pial mesothelium and subpial vascular microstroma expressed c-erbB2/HER-2, CK7 and VEGFR-2. SCs/TCs neighbouring the neuronoglial units (NGUs) also expressed HER-2, which suggests a pial origin. These cells were also positive for CD10, CD31, CD34, CD68 and nestin. Endothelial cells expressed CD10, CD31, CD34, CD146, nestin and vWF. We also found vasculogenic networks with spindle-shaped and stellate endothelial progenitors expressing CD10, CD31, CD34, CD68, CD146 and VEGFR-2. Isolated mesenchymal stromal cells expressed Stro-1, CD146, CK7, c-kit and nestin. Pericytes expressed α-SMA and CD146. Using transmission electron microscopy (TEM), we found endothelial-specific Weibel-Palade bodies in spindle-shaped stromal progenitors. Our study supports the hypothesis that an intrinsic vasculogenic niche potentially involved in microvascular maintenance and repair might be present in the human adult trigeminal ganglion and that it might be supplied by either the pial mesothelium or the bone marrow niche., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8 EGFPf/+ ) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Accumulating evidence has demonstrated a possible role of mitochondrial dysfunction in migraine pathophysiology. Migraine sufferers exhibit impaired metabolic capacity, with an increased formation of reactive oxygen species (ROS). Mitochondrial dynamics and mitochondrial biogenesis are key processes regulating mitochondrial homeostasis. The aim of this study was to explore the alterations of mitochondrial regulatory networks in a rat model of migraine induced by repeated dural stimulation with inflammatory soup (IS). Ultrastructural, protein, gene and mitochondrial DNA analysis were applied to assess mitochondrial dynamics and biogenesis in trigeminal ganglion (TG) neurons. Mitochondria in TG neurons exhibited small and fragmented morphology after repeated dural stimulation. Further investigations showed that mitochondrial fission protein dynamin-related protein 1 (Drp1) was increased while mitochondrial fusion protein Mitofusin1 (Mfn1) was reduced in TG neurons. In addition, our results also presented that mitochondrial DNA copy number in TG neurons was reduced significantly, accompanied by alterations in mRNA and protein levels of regulatory factors related to mitochondrial biogenesis including peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1α) and its downstream regulators in TG neurons in the IS-induced migraine model. These findings suggest that the mitochondrial dynamic regulatory networks are maladjusted in TG neurons in a rat model of migraine. Regulation of mitochondrial dynamics and biogenesis signaling may indicate a new mitochondria-targeted therapeutic strategy for migraine., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Increasing evidence shows that the homomeric glycine receptor is expressed in axon terminals and is involved in the presynaptic modulation of transmitter release. However, little is known about the expression of the glycine receptor, implicated in the presynaptic modulation of sensory transmission in the primary somatosensory neurons and their central boutons. To address this, we investigated the expression of glycine receptor subunit alpha 3 (GlyRα3) in the neurons in the trigeminal ganglion and axon terminals in the 1st relay nucleus of the brainstem by light- and electron-microscopic immunohistochemistry. Trigeminal primary sensory neurons were GlyRα3-immunopositive/gephyrin-immunonegative (indicating homomeric GlyR), whereas GlyRα3/gephyrin immunoreactivity (indicating heteromeric GlyR) was observed in dendrites. GlyRα3 immunoreactivity was also found in the central boutons of primary afferents but far from the presynaptic site and in dendrites at subsynaptic sites. Boutons expressing GlyRα3 contained small round vesicles, formed asymmetric synapses with dendrites and were immunoreactive for glutamate. These findings suggest that trigeminal primary afferent boutons receive presynaptic modulation via homomeric, extrasynaptic GlyRα3, and that different subtypes of GlyR may be involved in pre- and postsynaptic inhibition.

Neurons from sensory ganglia are exposed to oxidative attack in diabetes. Altered mitochondrial morphologies are due to impaired dynamics (fusion, fission) and to cristae remodeling. This study aimed to evaluate using transmission electron microscopy mitochondrial changes in diabetic trigeminal ganglia suggestive for ignition of apoptosis, in absence of "classical" morphological signs of apoptosis. We used samples of trigeminal ganglia (from six type 2 diabetes human donors and five streptozotocin (STZ)-induced diabetic rats). In human diabetic samples we found three main distributions of mitochondria: (a) small "dark" normal mitochondria, seemingly resulted from fission processes; (b) small "dark" damaged mitochondria, with side-vesiculations (single- and double-coated), large matrix vesicles and cytosolic leakage of reactive species, mixed with larger "light" mitochondria, swollen, and with crystolysis; (c) prevailing "light" mitochondria. In STZ-treated rats a type (c) distribution prevailed, except for nociceptive neurons where we found a different distribution: large and giant mitochondria, suggestive for impaired mitochondrial fission, mitochondrial fenestrations, matrix vesicles interconnected by lamellar cristae, and mitochondrial leakage into the cytosol. Thus, the ultrastructural pattern of mitochondria damage in diabetic samples of sensory neurons may provide clues on the initiation of intrinsic apoptosis, even if the classical morphological signs of apoptosis are not present. Further studies, combining use of biochemical and ultrastructural techniques, may allow a better quantification of the degree in which mitochondrial damage, with membrane alterations and cytosolic leaks, may be used as morphological signs suggesting the point-of-no return for apoptosis. Anat Rec, 299:1561-1570, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Telocytes (TCs) are typically defined as cells with telopodes by their ultrastructural features. Their presence was reported in various organs, however little is known about their presence in human trigeminal ganglion. To address this issue, samples of trigeminal ganglia were tested by immunocytochemistry for CD34 and examined by transmission electron microscopy (TEM). We found that TCs are CD34 positive and form networks within the ganglion in close vicinity to microvessels and nerve fibers around the neuronal-glial units (NGUs). TEM examination confirmed the existence of spindle-shaped and bipolar TCs with one or two telopodes measuring between 15 to 53 μm. We propose that TCs are cells with stemness capacity which might contribute in regeneration and repair processes by: modulation of the stem cell activity or by acting as progenitors of other cells present in the normal tissue. In addition, further studies are needed to establish if they might influence the neuronal circuits.

The etiology of trigeminal neuropathic pain is not clear, but there is evidence that demyelination, expression of cytokines, neuropeptides, and neurotrophic factors are crucial contributors. In order to elucidate mechanisms underlying trigeminal neuropathic pain, we evaluated the time course of morphological changes in myelinated and unmyelinated trigeminal nerve fibers, expression of cytokine IL-1β, neuropeptide substance P (SP), nerve growth factor (NGF), and glial derived neurotrophic factor (GDNF) in peripheral and ganglion tissues, using a rat model of trigeminal neuropathic pain. Chronic constriction injury (CCI) of the infraorbital nerve (IoN), or a sham surgery, was performed. Mechanical allodynia was evaluated from day 3 to day 15 post-surgery. Trigeminal nerves were divided into 2 sections - distal to CCI and ganglion - for morphological analyses, immunohistochemistry (IL-1β, SP), and protein quantification by ELISA (NGF, GDNF). At early postoperative time points, decreased mechanical responses were observed, which were associated with demyelination, glial cell proliferation, increased immunoexpression of IL-1 β and SP, and impaired GDNF production. In the late postoperative period, mechanical allodynia was present with partial recovery of myelination, glial cell proliferation, and increased immunoreactivity of IL-1β and SP. Our data show that demyelination/remyelination processes are related to the development of pain behavior. IL-1β may have effects both in ganglia and nerves, while SP may be an important mediator at the nerve endings. Additionally, low levels of GDNF may produce impaired signaling, which may be involved in generation of pain., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and 2 (HCN2) are abundantly expressed in primary sensory neurons and contribute to neuronal excitability and pathological pain. We studied the expression of HCN1 and HCN2 in the rat trigeminal ganglion (TG) neurons and axons in the dental pulp, and the changes in their expression following inflammation, using light- and electron-microscopic immunocytochemistry and quantitative analysis. HCN1 and HCN2 were expressed predominantly in large-sized, neurofilament 200-immunopositive (+) or parvalbumin+ soma in the TG whereas they were expressed mostly in unmyelinated and small myelinated axons in the sensory root. The expression was particularly strong along the plasma membrane in the soma. In the dental pulp, majority of HCN1+ and HCN2+ axons coexpressed calcitonin gene-related peptide. They were expressed mainly in the peripheral pulp and pulp horn where the axons branch extensively in the dental pulp. The expression of HCN1 and HCN2 in TG neurons increased significantly in rats with experimentally induced inflammation of the dental pulp. Our findings support the notion that HCN1 and HCN2 are expressed mainly by both the soma of mechanosensitive neurons in the TG and peripheral axons of nociceptive neurons in the sensory root, and may play a role in the mechanisms of inflammatory pain from the dental pulp., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

The sensory innervation of the dental mesenchyme is essential for tooth function and protection. Sensory innervation of the dental pulp is mediated by axons originating from the trigeminal ganglia and is strictly regulated in time. Teeth can develop from cultured re-associations between dissociated dental epithelial and mesenchymal cells from Embryonic Day 14 mouse molars, after implantation under the skin of adult ICR mice. In these conditions however, the innervation of the dental mesenchyme did not occur spontaneously. In order to go further with this question, complementary experimental approaches were designed. Cultured cell re-associations were implanted together with trigeminal ganglia for one or two weeks. Although axonal growth was regularly observed extending from the trigeminal ganglia to all around the forming teeth, the presence of axons in the dental mesenchyme was detected in less than 2.5% of samples after two weeks, demonstrating a specific impairment of their entering the dental mesenchyme. In clinical context, immunosuppressive therapy using cyclosporin A was found to accelerate the innervation of transplanted tissues. Indeed, when cultured cell re-associations and trigeminal ganglia were co-implanted in cyclosporin A-treated ICR mice, nerve fibers were detected in the dental pulp, even reaching odontoblasts after one week. However, cyclosporin A shows multiple effects, including direct ones on nerve growth. To test whether there may be a direct functional relationship between immunomodulation and innervation, cell re-associations and trigeminal ganglia were co-implanted in immunocompromised Nude mice. In these conditions as well, the innervation of the dental mesenchyme was observed already after one week of implantation, but axons reached the odontoblast layer after two weeks only. This study demonstrated that immunodepression per se does stimulate the innervation of the dental mesenchyme.

Headaches can be evoked by activation of meningeal nociceptors, but an involvement of pericranial tissues is debated. We aimed to examine a possible extracranial innervation by meningeal afferents in the rat. For in vivo neuronal tracing, dextran amines were applied to the periosteum underlying the temporal muscle. Labeling was observed 2 days later in the parietal dura mater, trigeminal ganglion, and spinal trigeminal nucleus with confocal and electron microscopy. In the hemisected rat head, extracellular recordings were made from meningeal nerve fibers. Release of calcitonin gene-related peptide (CGRP) from the cranial dura mater during noxious stimulation of pericranial muscles was quantified. In vivo capsaicin was injected into the temporal muscle while meningeal blood flow was recorded. In the parietal dura mater, labeled C- and Aδ fibers ramified extensively, accompanied the middle meningeal artery, and passed through the spinosus nerve into the maxillary and mandibular, but not the ophthalmic division of the trigeminal ganglion. Some fibers could be traced into the ipsilateral spinal trigeminal nucleus. Electrophysiological recordings revealed afferent fibers with mechanosensitive receptive fields both in the dura mater and in the parietal periosteum. Noxious stimulation of the temporal muscle caused CGRP release from the dura mater and elevated meningeal blood flow. Collaterals of meningeal nerve fibers project through the skull, forming functional connections between extra- and intracranial tissues. This finding offers a new explanation of how noxious stimulation of pericranial tissues can directly influence meningeal nociception associated with headache generation and why manual therapies of pericranial muscles may be useful in headaches., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Varicella zoster virus (VZV) latency is characterized by limited virus gene expression and the absence of virus DNA replication. Investigations of VZV latency and reactivation have been hindered by the lack of an in vitro model of virus latency. Since VZV is an exclusively human pathogen, we used naturally infected human trigeminal ganglia (TG) obtained at autopsy to study virus latency. Herein, we report optimization of medium to maintain TG integrity as determined by histology and immunohistochemistry. Using the optimized culture medium, we also found that both herpes simplex virus-1 (HSV-1) and VZV DNA replicated in TG explants after 5 days in culture. The increase in HSV-1 DNA was fourfold greater than the increase in VZV DNA. Overall, we present a model for alphaherpesvirus latency in human neurons in which the key molecular events leading to virus reactivation can be studied.

Integumentary sensory organs (ISOs) are densely distributed on the jaws of crocodilians and on body scales of members of the families Crocodilidae and Gavialidae. We examined the distribution, anatomy, innervation and response properties of ISOs on the face and body of crocodilians and documented related behaviors for an alligatorid (Alligator mississippiensis) and a crocodylid (Crocodylus niloticus). Each of the ISOs (roughly 4000 in A. mississippiensis and 9000 in C. niloticus) was innervated by networks of afferents supplying multiple different mechanoreceptors. Electrophysiological recordings from the trigeminal ganglion and peripheral nerves were made to isolate single-unit receptive fields and to test possible osmoreceptive and electroreceptive functions. Multiple small (<0.1 mm(2)) receptive fields, often from a single ISO, were recorded from the premaxilla, the rostral dentary, the gingivae and the distal digits. These responded to a median threshold of 0.08 mN. The less densely innervated caudal margins of the jaws had larger receptive fields (>100 mm(2)) and higher thresholds (13.725 mN). Rapidly adapting, slowly adapting type I and slowly adapting type II responses were identified based on neuronal responses. Several rapidly adapting units responded maximally to vibrations at 20-35 Hz, consistent with reports of the ISOs' role in detecting prey-generated water surface ripples. Despite crocodilians' armored bodies, the ISOs imparted a mechanical sensitivity exceeding that of primate fingertips. We conclude that crocodilian ISOs have diverse functions, including detection of water movements, indicating when to bite based on direct contact of pursued prey, and fine tactile discrimination of items held in the jaws.

We present a case of a rare tumor--melanotic schwannoma of trigeminal nerve root and gasserian ganglion. Differential diagnosis between metastatic melanoma and melanotic schwannoma (MS) is associated with serious difficulties and high responsibility. Metastatic melanoma is a high grade tumor while most MS are benign lesions with good outcome. By the date 105 cases of these tumors are described in the world literature, 3 of them originated from trigeminal nerve root and gasserian ganglion. MS predominantly occur in relatively young patients, they are characterized by presence of Carney's complex and psammomatous bodies and absence of primary focus. MS and metastatic melanoma have similar appearance on MRI due to presence of melanin granules. Indirect signs evident for MS include cystic structure and dumbbell-shaped growth. Metastatic melanoma of cranial nerves is more typical in people older than 40, primary focus in the face in the zone of innervation of affected nerve is common. In case of absence of the listed features differential diagnosis is based on immunohistochemical analysis and electron microscopy of tissue samples.

Previous studies reported that a subpopulation of mouse and rat trigeminal neurons express water channel aquaporin-1 (AQP1). In this study we make a comparative investigation of AQP1 localization in the human and mouse trigeminal systems. Immunohistochemistry and immunofluorescence results showed that AQP1 was localized to the cytoplasm and cell membrane of some medium and small-sized trigeminal neurons. Additionally, AQP1 was found in numerous peripheral trigeminal axons of humans and mice. In the central trigeminal root and brain stem, AQP1 was specifically expressed in astrocytes of humans, but was restricted to nerve fibers within the central trigeminal root and spinal trigeminal tract and nucleus in mice. Furthermore, AQP1 positive nerve fibers were present in the mucosal and submucosal layers of human and mouse oral tissues, but not in the muscular and subcutaneous layers. Fluorogold retrograde tracing demonstrated that AQP1 positive trigeminal neurons innervate the mucosa but not skin of cheek. These results reveal there are similarities and differences in the cellular localization of AQP1 between the human and mouse trigeminal systems. Selective expression of AQP1 in the trigeminal neurons innervating the oral mucosa indicates an involvement of AQP1 in oral sensory transduction.

Objectives: The presence of c-kit positive neurons in sensory ganglia has been verified in various species but not in humans. Our aim has been to identify whether human primary trigeminal neurons label with c-kit/CD117 and thus, whether data gathered in animal studies can be extrapolated to humans. We also intended to establish whether, and which non-neuronal cells also label with c-kit in the trigeminal ganglion., Methods: Human adult trigeminal ganglia from eight cadavers were processed for immunohistochemistry on paraffin embedded samples using monoclonal antibodies for CD117/c-kit, and three additional trigeminal ganglia were used for transmission electron microscopy (TEM). To evaluate which neuronal type (A or B) was labeled with c-kit, we evaluated the same neurons on adjacent sections labeled with antibodies for neurofilaments (NF)., Results: c-kit has labeled trigeminal neurons (TNs), mast cells and interstitial cells (ICs) within the trigeminal ganglion. c-kit+TNs were NF-and thus were strongly presumed to be nociceptive, as such neurons are known to be NF-poor. c-kit+ICs with long and moniliform processes intermingled with the satellite glial cells (SGCs) of the neuronal envelopes. TEM evaluations confirmed this mixed composition of the neuronal envelopes and demonstrated that the perineuronal ICs are in fact interstitial Cajal-like cells (ICLCs) and/or telocytes., Conclusions: c-kit+TNs were objectified in humans and strongly presumed to be nociceptive. TNs envelopes mostly consist of SGCs, but are also combined with ICLCs/telocytes., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Glypicans are conserved cell surface heparan sulfate proteoglycans expressed in a spatiotemporally regulated manner in many developing tissues including the nervous system. Here, we show that Glypican-1 (GPC1) is expressed by trigeminal placode cells as they ingress and contribute to trigeminal sensory neurons in the chick embryo. Either expression of full-length or truncated GPC1 in vivo causes defects in trigeminal gangliogenesis in a manner that requires heparan sulfate side chains. This leads to either abnormal placodal differentiation or organization, respectively, with near complete loss of the ophthalmic (OpV) trigeminal ganglion in the most severe cases after overexpression of full-length GPC1. Interestingly, modulating GPC1 alters levels of endogenous Wnt signaling activity in the forming trigeminal ganglion, as indicated by Wnt reporter expression. Accordingly, GPC1 overexpression phenocopies Wnt inhibition in causing loss of OpV placodal neurons. Furthermore, increased Wnt activity rescues the effects of GPC1 overexpression. Taken together, these results suggest that appropriate levels of GPC1 are essential for proper regulation of canonical Wnt signaling during differentiation and organization of trigeminal placodal cells into ganglia., (Copyright © 2010. Published by Elsevier Inc.)

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in adult sensory neuron function. However, the acute effects of GDNF on primary sensory neuron excitability remain to be elucidated. The aim of the present study was to investigate whether GDNF acutely modulates the excitability of adult rat trigeminal ganglion (TRG) neurons that innervate the facial skin by using perforated-patch clamping, retrograde-labeling and immunohistochemistry techniques. Fluorogold (FG) retrograde labeling was used to identify the TRG neurons innervating the facial skin. The FG-labeled small- and medium-diameter GDNF immunoreactive TRG neurons, and most of these neurons also expressed the GDNF family receptor alpha-1 (GFRalpha-1). In whole-cell voltage-clamp mode, GDNF application significantly inhibited voltage-gated K(+) transient (I(A)) and sustained (I(K)) currents in most dissociated FG-labeled small-diameter TRG neurons. This effect was concentration-dependent and was abolished by co-application of the protein tyrosine kinase inhibitor, K252b. Under current-clamp conditions, the repetitive firing during a depolarizing pulse were significantly increased by GDNF application. GDNF application also increased the duration of the repolarization phase and decreased the duration of the depolarization phase of the action potential, and these characteristic effects were also abolished by co-application of K252b. These results suggest that acute application of GDNF enhances the neuronal excitability of adult rat small-diameter TRG neurons innervating the facial skin, via activation of GDNF-induced intracellular signaling pathway. We therefore conclude that a local release of GDNF from TRG neuronal soma and/or nerve terminals may regulate normal sensory function, including nociception.

Objective: To study the distribution character of the trigeminal sensory afferent fibers in rat's trigeminal sensory root (RxV) and to explore the possibility of selectively injury of the pain afferent fibers., Methods: The retrograde tracer fluorogold (FG) was injected into trigeminal spinal subnucleus and pontine nucleus respectively. After 7 days survival, the rats were sacrificed and the RxV was removed and sectioned. The distribution of FG at RxV was studied under fluorescent microscope and its character was analyzed., Results: After being introduced into trigeminal spinal rostral nucleus, the FG could be observed at the lateral portion on the sections of RxV. The fibers were small and concentrated. The interpolaris subnucleus and caudal subnucleus injection group also showed small and concentrated fibers in the lateral portion of RxV, but the distribution area was larger than that of the rostral subnucleus group. While being injected into trigeminal pontine nucleus, the FG positive axons could be found in the ventral, medial and central portion of the RxV cross sections. These fibers were thicker and more scattered compared to those projecting into trigeminal spinal nucleus., Conclusion: The fibers relating to transporting pain sensory concentrate at a certain area of rat' s RxV, which indicate that selectively injury of the pain afferent fibers of RxV should be possible.

Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including alpha-mannosidosis. Here we show that the neuropathology of a mouse model for alpha-mannosidosis can be efficiently treated using recombinant human alpha-mannosidase (rhLAMAN). After intravenous administration of different doses (25-500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (<70%) in visceral tissues, higher doses were needed for a clear effect in central and peripheral nervous tissues. A distinct reduction (<50%) of brain storage required repeated high-dose injections (500 U/kg), whereas lower doses (250 U/kg) were sufficient for clearance of stored substrates in peripheral neurons of the trigeminal ganglion. Successful transfer across the blood-brain barrier was evident as the injected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storage vacuoles. Importantly, the decrease in neuronal storage in the brain correlated with an improvement of the neuromotor disabilities found in untreated alpha-mannosidosis mice. Uptake of rhLAMAN seems to be independent of mannose-6-phosphate receptors, which is consistent with the low phosphorylation profile of the enzyme. These data suggest that high-dose injections of low phosphorylated enzymes might be an interesting option to efficiently treat LSDs with CNS involvement.

Trigeminal primary afferents expressing P2X(3) 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 P2X(3)-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, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) 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 P2X(3) (+) 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 P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN., ((c) 2007 Wiley-Liss, Inc.)

Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity.

In this study we have used the transcription assay with 5'-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5'-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5'-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5'-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5'-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5'-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5'-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems.

A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.

Previous studies have shown that during acute infection of the porcine trigeminal ganglia (TG), Aujeszky's disease virus (ADV)-infected neurons are protected from apoptosis induced by the virus itself and by cells of the immune system. However, TG neurons productively infected by ADV finally die and are phagocytosed by adjacent cells, a fact that leads us to speculate that the inhibition of neuronal apoptosis by ADV may be temporary rather than absolute. To address this issue we used TG and brain stem from pigs during acute infection by ADV. Infected cells were detected by immunohistochemical staining of viral antigens, whereas apoptotic cells were identified with an anti-active caspase-3 antibody, the TUNEL assay and by transmission electron microscopy. The results obtained in this study support the contention that the inhibition of neuronal apoptosis by ADV is temporary, since activation of caspase-3 could be detected in infected neurons at late stages in infection and because foci of advanced neuronophagia contained neurons exhibiting typical ultrastructural features of apoptosis.

In classical chromatolysis, Nissl bodies are initially lost centrally with subsequent progression to the periphery of the neuron. Peripheral chromatolysis has the opposite pattern; it is less common and more difficult to produce. We describe a new method for producing peripheral chromatolysis in neurons of trigeminal ganglia and dorsal root ganglia that requires only injection of large doses of lithium chloride (LiCl) for two, three or four consecutive daily doses. This method may be useful for elucidating the intraneuronal mechanisms that control the location and structure of the Nissl bodies.

An ultrastructural study was performed on trigeminal ganglia removed from foetuses aged 10 to 23 weeks. The first turns of lemmocyte processes around the axons were observed in the trigeminal ganglion in the 12th week of development. During the period between the 15th and the 18th weeks the myelin sheath increases in thickness and becomes a compact, laminated structure. In foetuses of 23 weeks the compact myelin sheath has up to 24 myelin lamellae.

Nodes of Ranvier in myelinated fibers exhibit a complex architecture in which specific molecules organize in distinct nodal, paranodal and juxtaparanodal domains to support saltatory conduction. The clustering of sodium channel Na(v)1.6 within the nodal membrane has led to its identification as the major nodal sodium channel in myelinated axons. In contrast, much less is known about the molecular architecture of nonmyelinated fibers. In the present study, Na(v)1.6 is shown to be a significant component of nonmyelinated PNS axons. In DRG C-fibers, Na(v)1.6 is distributed continuously from terminal receptor fields in the skin to the dorsal root entry zone in the spinal cord. Na(v)1.6 is also present in the nerve endings of corneal C-fibers. Analysis of compound action potential recordings from wildtype and med mice, which lack Na(v)1.6, indicates that Na(v)1.6 plays a functional role in nonmyelinated fibers where it contributes to action potential conduction. These observations indicate that Na(v)1.6 functions not only in saltatory conduction in myelinated axons but also in continuous conduction in nonmyelinated axons., (Copyright 2002 Elsevier Science B.V.)

We used transmission electron microscopic montages to examine the composition of nerve bundles serving the infrared pit organs of two species of crotaline snakes, Agkistrodon blomhoffii and A. brevicaudus. In the three main bundles, the myelinated fibers totaled 2,200-3,700, and unmyelinated fibers 2,400. We also discovered for the first time two accessory bundles composed almost entirely of unmyelinated fibers running alongside the main bundles, containing an average total of 3,300 unmyelinated fibers vs. an average of 10 myelinated fibers. Thus, the average total of unmyelinated fibers was nearly twice that of myelinated fibers. To study the nature of the unmyelinated fibers, we did double staining immunohistochemistry with antibodies for substance P (SP) and vasoactive intestinal polypeptide (VIP) in combination with and without capsaicin pretreatment. SP and VIP immunoreactive varicose fibers ran straight toward the center of the pit membrane in parallel with arterioles and venules, and also formed a dense network around the periphery of the membrane. There were three types of fibers: fibers containing only SP, fibers containing only VIP, and fibers containing both peptides. SP-only fibers were distributed singly throughout the pit membrane and in small bundles around the periphery. SP+VIP fibers were distributed sparsely in the pit membrane and around its periphery. VIP-only fibers were distributed throughout the pit membrane and were of smaller diameter than SP and SP+VIP fibers. After treatment with capsaicin, most of the three types of varicose fibers disappeared from the central part of the pit membrane, but those around the periphery remained unaffected. The capsaicin-sensitive fibers may be unmyelinated sensory types, and the unaffected ones may be autonomic nerve fibers., (Copyright 2002 Wiley-Liss, Inc.)

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.

The vanilloid receptor VR1 and the vanilloid receptor-like protein VRL-1 are associated with polymodal nociceptors, and may be important for pain processing in normal and injured teeth. Using immunohistochemistry, we have studied the distribution of these receptors in rat pulpal or gingival trigeminal ganglion neurons that were identified through retrograde labeling with fluoro-gold. Twenty-one percent to 34% of tooth pulp-innervating neurons were VRl-positive, while 32%-51% were VRL-1-immunoreactive. However, double-labeling experiments revealed that VR1 and VRL-1 rarely co-existed in the same cells, but rather seemed to be confined to separate subpopulations. Among the gingival neurons, about 25% were VR1-positive and about 41% were VRL-1-immunoreactive. A lesion of the inferior alveolar nerve, which supplies mandibular teeth and gingiva, resulted in a marked down-regulation of VR1 in the affected trigeminal ganglion cells. A down-regulation of VRL-l was also indicated. The results suggest that both VR1 and VRL-1 could have significant roles in pulpal and gingival nociceptive transduction.

Capsaicin acts on the vanilloid receptor subtype 1, a noxious heat-gated cation channel located on a major subgroup of nociceptive primary afferent neurons. Following the systemic capsaicin treatment of neonatal rats, the loss of B-type sensory neurons in trigeminal ganglion of adult rats with chemoanalgesia and abolition of neurogenic inflammation was investigated. Our quantitative morphometric analysis revealed that in the trigeminal ganglion of neonatal rats treated with 50 mg/kg s.c. capsaicin, the total number of neurons, morphology of B-type cells and cell-size histograms did not differ from that of the controls 1 or 5 days after treatment. These observations indicate that early cell death does not play a significant part in the loss of B-type cells, which in our sample was 39.4% on the 19th day. However under the electron microscope pronounced selective mitochondrial swelling with disorganized cristae was observed in B-type neurons at 1-20 weeks after capsaicin treatment. Daily treatment with nerve growth factor (NGF, 10 x 100 microg/kg s.c.), started 1 day after capsaicin injection, prevented the loss of B-type cells but did not counteract the development of long-lasting mitochondrial damage. After NGF treatment, partial restitution of chemonociception to capsaicin instillation into the eye occurred but capsaicin-induced inhibition of neurogenic plasma extravasation in the hindpaw evoked by topical application of mustard oil remained unaltered. We conclude, that capsaicin treatment in neonatal rats, as in the adults, destroys terminal parts of the sensory neurons supplied by vanilloid receptors and induces long-lasting mitochondrial swelling in the soma. We hypothesize that loss of NGF uptake results in delayed cell death of B-type neurons in neonates., (Copyright 2002 IBRO)

We investigated the effects of percutaneous gasserian glycerol injection in dogs and reviewed the histopathological changes. Experiments were performed in 16 adult healthy mongrel dogs. In group 1 (8 dogs) normal saline and in group 2 (8 dogs) pure glycerol was injected in the right trigeminal ganglion. After these procedures, dogs in each group were sacrificed after 24 h (3 dogs), 7 days (3 dogs), 21 days (2 dogs). The trigeminal ganglion and nerve of both sides were removed by using microsurgical techniques and examined by light and electron microscopy. Group 1: in all sections, nerve cells, myelinated and nonmyelinated fibers revealed normal patterns with slight fibrosis. Group 2: in all sections, myelinated fibers showed disintegration and swelling of the myelin sheath, rupture of axon continuity, destruction of basal lamina, deformation of the myelin-axon relationship by both light microscopy and electron microscopy. The sections examined by electron microscopy also showed axonolysis in nonmyelinated fibers. The changes after 7 and 21 days were less prominent than after 24 h. In the left sides, there are no pathological changes. Glycerol has a neurolytic effect on the dog's trigeminal ganglion. These effects were not specific and selective for myelinated and nonmyelinated nerve fibers., (Copyright 2002 S. Karger AG, Basel)

The expression in various cell types of peripheral tissues of glial fibrillary acidic protein (GFAP), first discovered as an intermediate filament specific for astrocytes, remains controversial owing to numerous reports of a wide distribution for GFAP-immunoreactivity in various cells. The present study employed immunohistochemistry to investigate the precise expression of GFAP in the dental pulp and trigeminal ganglion of adult rats and wild-type mice as well as GFAP-knockout mice. The exhibition of GFAP-immunoreactivity in the trigeminal ganglion was further examined by a reverse transcription polymerase chain reaction (RT-PCR) technique, and in situ hybridization histochemistry using a specific cRNA probe prepared by us. The immunoreaction for GFAP was recognizable in the axons, Schwann cells, and the fibroblasts in the dental pulp of rats and wild-type littermate mice. However, mice with null mutations in the GFAP gene remained immunoreactive for GFAP in all these locations. Intense GFAP-immunoreactivity was found in a small number of satellite cells in the trigeminal ganglion in all animals examined in this study. RT-PCR analysis demonstrated bands for the GFAP gene corresponding to the length expected from the primer design in the samples of trigeminal ganglion and dental pulp. In situ hybridization histochemistry also showed intense signals for GFAP mRNA in some satellite cells of the trigeminal ganglion, but never in the neurons. These data suggest that the GFAP-immunoreactive molecules in the pulpal axons and fibroblasts react non-specifically with the polyclonal antibody and are probably a closely related type of intermediate filament.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents., (Copyright 2001 Wiley-Liss, Inc.)

Possible roles of the US3 gene of the herpes simplex virus (HSV) in the interaction between the virus and primary afferent neurons were examined. Neuronal apoptosis was observed in the trigeminal ganglion of mice that were infected with the wild-type (wt) of HSV-2 strain 186 and with US3-deficient mutant virus (L1BR1). In wt virus-infected mice, many HSV-immunoreactive (HSV-ir) cells were seen throughout the trigeminal ganglion, although no apoptotic change was detected. On the other hand, HSV-ir cells in L1BR1-infected mice were found only in the ophthalmic division of the trigeminal ganglions. Examination by HSV-immunohistochemistry combined with the terminal deoxynucleotidal transferase (Tdt)-mediated deoxyuridin 5'-triphosphate (dUTP) nick-end labeling (TUNEL) method showed that DNA fragmentation had occurred in almost all HSV-ir cells in the L1BRI-infected ganglion. Ultrastructurally, many viral particles were detected in apoptotic ganglionic neurons of mice infected with L1BR1. These results indicate that US3 protein kinase (US3pk) played a role in protecting HSV-infected primary afferent neurons from apoptotic cell death. The present study suggests that US3pk plays a role when HSV establishes latent infections in the sensory ganglia.

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.

Sympathetic axons invade the trigeminal ganglia of mice overexpressing nerve growth factor (NGF) (NGF/p75(+/+) mice) and surround sensory neurons having intense NGF immunolabeling; the growth of these axons appears to be directional and specific (). In this investigation, we provide new insight into the neurochemical features and receptor requirements of this sympathosensory sprouting. Using double-antigen immunohistochemistry, we demonstrate that virtually all (98%) trigeminal neurons that exhibit a sympathetic plexus are trk tyrosine kinase receptor (trkA)-positive. In addition, the majority (86%) of those neurons enveloped by sympathetic fibers is also calcitonin gene-related peptide (CGRP)-positive; a smaller number of plexuses (14%) surrounded other somata lacking this neuropeptide. Our results show that sympathosensory interactions form primarily between noradrenergic sympathetic efferents and the trkA/CGRP-expressing sensory somata. To assess the contribution of the p75 neurotrophin receptor (p75(NTR)) in sympathosensory sprouting, a hybrid strain of mice was used that overexpresses NGF but lacks p75(NTR) expression (NGF/p75(-/-) mice). The trigeminal ganglia of NGF/p75(-/-) mice, like those of NGF/p75(+/+) mice, have increased levels of NGF protein and display a concomitant ingrowth of sympathetic axons. In contrast to the precise pattern of sprouting seen in the ganglia of NGF/p75(+/+) mice, sympathetic axons course randomly throughout the ganglionic neuropil of NGF/p75(-/-) mice, forming few perineuronal plexuses. Our results indicate that p75(NTR) is not required to initiate or sustain the growth of sympathetic axons into the NGF-rich trigeminal ganglia but rather plays a role in regulating the directional patterns of axon growth.

Single gustatory nerve fibers branch and innervate several taste buds. In turn, individual taste buds may receive innervation from numerous gustatory nerve fibers. To evaluate the pattern of sensory innervation of fungiform papilla-bearing taste buds, we used iontophoretic fluorescent injection to retrogradely label the fibers that innervate single taste papillae in the hamster. For each animal, a single taste papilla was injected through the gemmal pore with 3.3% tetramethylrhodamine dextran amine. Fungiform papillae either at the tongue tip (0.5-1.5 mm from the tip) or more posteriorly (1.5-3.0 mm from the tip) were injected. After one to seven days survival, the geniculate and trigeminal ganglia and the tongue were sectioned and examined for labeled cells and fibers, respectively. Analysis of the number and topographic distribution of geniculate cells innervating single taste papillae, shows that: (i) 15 +/- 4 (S.D.) ganglion cells converge to innervate a single fungiform taste bud; (ii) more ganglion cells innervate anterior- (range: 13-35 cells) than posterior-lying buds (range: five to 12 cells), which, in part, may be related to bud volume (microm3); and (iii) ganglion somata innervating a single taste bud are scattered widely within the geniculate ganglion. Analysis of labeled fibers in the tongue demonstrated that two to eight taste buds located within 2 mm of the injected taste bud share collateral innervation with the injected taste bud. Since all buds with labeled fibers were located in close proximity (within a 2-mm radius), widely dispersed geniculate ganglion cells converge to innervate closely spaced fungiform taste buds. Trigeminal ganglion (mandibular division) cells were also labeled in every case and, as with the geniculate ganglion, a dispersed cell body location and collateralization pattern among papillae were observed. This study shows that iontophoresis of tetramethylrhodamine dextran amine, selectively applied to individual peripheral receptor end-organs, effectively locates sensory ganglion cells in two different ganglia that project to these sites. Moreover, the marker demonstrates collateral branches of sensory afferents associated with the labeled fibers and the nearby receptor areas innervated by these collaterals. The labeling of single or clusters of receptor cells, as well as identified sensory afferents, affords future possibilities for combining this technique with immunocytochemistry to establish the relationships of innervation patterns with neurotransmitters and neurotropic substances within identified cells.

Previous work suggested that in mouse, presumptive targets of the trigeminal ganglion, rather than intermediate structures, attract pioneer axons from the time their growth cones exit the ganglion (Lumsden and Davies, 1986). In rat we find that some presumptive targets repel trigeminal axons. The repellant activity is concentrated in the anterior and ventral epithelium of the mandibular arch at embryonic day 12 (E12) and was also present in the maxillary arch. The activity is blocked by anti-neuropilin-1. E13 mandible explants repel trigeminal axons during the first day of outgrowth in vitro, but thereafter permit or attract trigeminal ganglion axon outgrowth. By E14, lingual nerve afferents first enter the tongue in vivo, and the repellant influence becomes restricted to the midline. The progressive restriction of the repellant influence may contribute to the in vivo progression of nerve development: the earliest afferents turn anteriorly lateral to the tongue, but subsequently arriving afferents advance into the tongue and then turn away from the midline. Thus, the repellant may influence the order of nerve branch development and the timing of innervation of epithelial and subepithelial targets. Heterochronic studies revealed that the loss of repellant influence from presumptive lateral tongue surface results from downregulation of the repellant activity, not of responsiveness to the repellant. Because presumptive targets repel trigeminal axons during the initial stages of advance from the trigeminal ganglion and do not have a net attractive influence until after afferents have arrived near the target, intermediate structures must guide these axons initially.

The cells of origin of peripheral nerves that supply the temporomandibular joint were investigated by examining the centripetal transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Following WGA-HRP injection into the temporomandibular joint capsule of the cat, a large number of labelled neurones were observed in the trigeminal and superior cervical ganglia ipsilateral to the injection site, while no labelled neurones were detected in the cervical dorsal-root ganglia. Only one labelled neurone was seen in the stellate ganglion. Labelled neurones were primarily located in the posterolateral and dorsal regions of the trigeminal ganglion, but their distribution in the superior cervical ganglion was not localized to specific regions. The labelled neurones in the trigeminal ganglion were significantly larger than those in the superior cervical ganglion but the sizes of smaller neurones overlapped, suggesting that trigeminal ganglion neurones send both myelinated and unmyelinated fibres to the temporomandibular joint. The innervation of the temporomandibular joint by somatosensory and sympathetic fibres suggests that sympathetic nerves could be responsible for allodynia or neuropathic pain caused by temporomandibular disorders.

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.

Vibrissa connections play a decisive role in setting the somatotopic coordinates in the trigeminal sensory system. Although previous studies have examined the development of peripheral patterning, certain questions are still in dispute, for example, the way vibrissa connections are structured, and the relationship between periphery and central organization, and ganglion cell organization. In order to fill the blanks left by previous studies, the extension of ganglionic branches and the formation of vibrissa connections were reexamined by using fluorescent carbocyanin dyes, DiI and DiA, during embryonic days 10 to 14 in mouse. Whole-mount preparations satisfactorily demonstrated the ganglionic fiber system, which allowed detailed analysis at both macroscopic and microscopic levels. We show here that the differential fasciculation of follicular nerves is the critical process for organizing vibrissa connections which modulate the initially extending fiber pattern. Follicular nerves developed by fine fibers arising from initially ordered root fascicles, so as to connect with vibrissa rudiments that developed on the facial prominences. During differential fasciculation, ganglion cells were segregated into distinct groups by the vibrissa connections, whereas central fiber terminals did not yet develop specific structures in the nuclear region. In the primary order of the trigeminal sensory system, vibrissa connections in the periphery were organized before those of the central structure. These results indicate that trigeminal ganglion cells have a critical binomial function in order to transfer the somatotopic relations among vibrissa rudiments into the topographic coordinates of the central system.

The effect of this axotomy on the expression of vasoactive intestinal polypeptide (VIP) in trigeminal ganglion neurones and nerve fibres in the first molar was examined immunohistochemically 3 weeks postsurgically in rats. A distinct upregulation of VIP-like immunoreactivity was found in 3 to 4% of the neurones (mean dia., 20.9 +/- 0.45 microns; mean cross-sectional area, 367 +/- 13.2 microns 2) in the mandibular region after axotomy. An almost complete coexpression was registered in neurones upregulated for VIP and growth-associated protein 43. Coexpression of VIP and substance P (SP) was found in a small number of the immunoreactive (IR) small-sized neurones, mainly in those in which VIP and SP were both weakly immunoreactive. In the uninjured ganglion, weakly labelled VIP-positive granules were frequently traced at high magnification in the cytoplasm of small neurones. No VIP-IR fibres were found in the control molar pulp, except for a few in the root pulp near the apex. However, 3 weeks after axotomy, a number of VIP-containing nerve fibres were found in the molar pulp and apical periodontium. Coarse VIP-IR fibres directed towards the odontoblast layer were a common finding. In some of these fibres VIP was shown to colocalize with SP. These results indicate that VIP is clearly expressed and transported in regenerating primary afferent neurones after axotomy of the inferior alveolar nerve.

Purpose: To characterize neuropeptide distribution in the ciliary ganglion of rhesus monkeys (Macaca mulatta)., Methods: Cryostat tissue sections of fixed rhesus monkey ciliary, pterygopalatine, superior cervical, and trigeminal ganglia were incubated with antisera to neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Antibody binding was visualized by indirect immunofluorescence., Results: NPY-like immunoreactive (LI) nerve terminals surrounded 80% of ciliary ganglion cells, but ciliary ganglion cell somata were unstained. NPY-LI cells were present in the superior cervical ganglion, in which almost all cells were TH- and DBH-LI, and in the pterygopalatine ganglion, in which almost all cells were VIP-LI. Because neither TH, DBH, nor VIP immunoreactivity was detected in nerves contacting ciliary ganglion cells, the NPY-LI input to ciliary neurons does not likely derive from the autonomic ganglia. The trigeminal ganglion, another potential source, had no NPY-LI neurons. CGRP- and SP-LI axons from the nasociliary nerve traversed the ciliary ganglion; a small number of varicose axons were distributed among ganglion cells and rarely surrounded cell somata. Most ciliary ganglion cells were TH-LI, but only a few were DBH-LI., Conclusions: Based on these patterns of peptide immunoreactivities, the NPY-LI nerve fibers investing ciliary ganglion cells in the rhesus monkey are most likely preganglionic axon terminals of mesencephalic parasympathetic neurons. Although the origin and function of these NPY-LI nerves remains to be established, the present finding adds to the remarkable diversity of neuropeptide immunoreactivity so far identified in preganglionic and postganglionic cells of the ciliary ganglion in different species of birds and mammals, including primates.

Nerve fibres were localized during the initiation and early morphogenesis of the first molar tooth in rat embryos by immunoperoxidase detection of the intermediate-filament protein peripherin and protein gene product 9.5 (PGP 9.5). Nerve fibres from the trigeminal ganglion were detected in the developing first branchial arch of E12-14 embryos. Nerves were not seen in the vicinity of the developing tooth germ before the buid stage (E15), when they were seen around the condensed dental mesenchyme. During transition from the bud to the cap stage (E15), nerve fibres were detected not only in the area of the future dental follicle but also in the mesenchyme next to dental epithelium on the buccal side of the tooth germ. During later cap and bell stages nerve fibres persisted in the dental follicle, but they were not seen in the epithelial dental organ or dental papilla mesenchyme. Absence of trigeminal nerve fibres from the presumptive tooth-bearing area indicates that they are not involved in the initiation of rat tooth development. In addition, the localization of nerve fibres shows that there are some differences in the innervation of rat teeth compared with human and mouse teeth. These results provide data for further studies on the regulation of embryonic rat tooth innervation.