Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Not Just an Anchor: The Human Filum Terminale Contains Stretch Sensitive and Nociceptive Nerve Endings and Responds to Electrical Stimulation With Paraspinal Muscle Activation.

BACKGROUND: Neural components of the fibrous filum terminale (FT) are well known but are considered as embryonic remnants without functionality. OBJECTIVE: To investigate the ultrastructure of human FT specimens for sensory nerve endings and record paraspinal muscle activity on electrostimulation of the FT. METHODS: We prospectively investigated a cohort of 53 patients who underwent excision of the FT for the treatment of tethered cord syndrome. Surgical FT specimens were investigated by light and transmission electron microscopy. Intraoperative electrophysiological routine monitoring was extended by recording paraspinal muscles above and below the laminotomy level. RESULTS: Light microscopy revealed tiny peripheral nerves piercing the pia mater of the FT and entering its fibrous core. Transmission electron microscopy unveiled within the fibrous core of the FT myelinated nerve structures in 8 of the 53 patients and unmyelinated ones in 10 of the 53 patients. Both nerve endings encapsulated in fibrous tissue or unencapsulated nonmyelinated Schwann cell nerve bundles, that is, Remak cells, were found. Those nerve endings resembled mechanoreceptor and nociceptive receptor structures found in human skin, muscle tendons, and skeletal ligaments. Specifically, we found Ruffini mechanoreceptors and in addition nerve endings which resembled nociceptive glioneural structures of the skin. Bipolar electrostimulation of the FT was associated with paraspinal muscle activity above and below the spinal segment at which the FT was stimulated. CONCLUSION: Morphological and electrophysiological results indicate the presence of functional sensory nerve endings in the FT. Like other spine ligaments, the FT may serve as a proprioceptive element but may also contribute to back pain in spine disorders.

Increased occurrence of pathological mitochondria in astrocytic perivascular endfoot processes and neurons of idiopathic intracranial hypertension.

Idiopathic intracranial hypertension (IIH) primarily affects fertile, overweight women, and presents with the symptoms of raised intracranial pressure. The etiology is unknown but has been thought to relate to cerebrospinal fluid disturbance or cerebral venous stenosis. We have previously found evidence that IIH is also a disease of the brain parenchyma, evidenced by alterations at the neurogliovascular interface, including astrogliosis, pathological changes in the basement membrane and pericytes, and alterations of perivascular aquaporin-4. The aim of this present electron microscopic study was to examine whether mitochondria phenotype was changed in IIH, particularly focusing on perivascular astrocytic endfeet and neurons (soma and pre- and postsynaptic terminals). Cortical brain biopsies of nine reference individuals and eight IIH patients were analyzed for subcellular distribution and phenotypical features of mitochondria using transmission electron microscopy. We found significantly increased prevalence of pathological mitochondria and reduced number of normal mitochondria in astrocytic endfeet of IIH patients. The degree of astrogliosis correlated negatively with the number of normal mitochondria in astrocytic endfoot processes. Moreover, we found significantly increased number of pathological mitochondria in pre- and postsynaptic neuronal terminals, as well as significantly shortened distance between mitochondria and endoplasmic reticulum contacts. Finally, the length of postsynaptic density, a marker of synaptic strength, was on average reduced in IIH. The present data provide evidence of pathological mitochondria in perivascular astrocytes endfeet and neurons of IIH patients, highlighting that impaired metabolism at the neurogliovascular interface may be a facet of IIH.

Distribution and morphology of P2X3-immunoreactive subserosal afferent nerve endings in the rat gastric antrum.

The present study investigated the morphological characteristics of subserosal afferent nerve endings with immunoreactivity for the P2X3 purinoceptor (P2X3) in the rat stomach by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3 immunoreactivity was observed in subserosal nerve endings proximal and lateral to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into several lamellar processes to form net-like complex structures that extended two-dimensionally in every direction on the surface of the longitudinal smooth muscle layer. The axon terminals in the periphery of P2X3-immunoreactive net-like structures were flat and looped or leaf-like in shape. Some net-like lamellar structures and their axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2. P2X3-immunoreactive nerve fibers forming net-like terminal structures were closely surrounded by S100B-immunoreactive terminal Schwann cells, whereas axon terminals twined around these cells and extended club-, knob-, or thread-like protrusions in the surrounding tissues. Furthermore, a retrograde tracing method using fast blue dye indicated that most of these nerve endings originated from the nodose ganglia of the vagus nerve. These results suggest that P2X3-immunoreactive subserosal nerve endings have morphological characteristics of mechanoreceptors and contribute to sensation of a mechanical deformation of the distal antral wall associated with antral peristalsis.

Palisade Endings Have an Exocytotic Machinery But Lack Acetylcholine Receptors and Distinct Acetylcholinesterase Activity.

Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

Glia-Neuron Interactions in Caenorhabditis elegans.

Glia are abundant components of animal nervous systems. Recognized 170 years ago, concerted attempts to understand these cells began only recently. From these investigations glia, once considered passive filler material in the brain, have emerged as active players in neuron development and activity. Glia are essential for nervous system function, and their disruption leads to disease. The nematode Caenorhabditis elegans possesses glial types similar to vertebrate glia, based on molecular, morphological, and functional criteria, and has become a powerful model in which to study glia and their neuronal interactions. Facile genetic and transgenic methods in this animal allow the discovery of genes required for glial functions, and effects of glia at single synapses can be monitored by tracking neuron shape, physiology, or animal behavior. Here, we review recent progress in understanding glia-neuron interactions in C. elegans. We highlight similarities with glia in other animals, and suggest conserved emerging principles of glial function.

Development of hair cell phenotype and calyx nerve terminals in the neonatal mouse utricle.

The vestibular organs of reptiles, birds, and mammals possess Type I and Type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a Type I or Type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in Type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse utricles between postnatal days 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to Type I hair cells) increased in all regions of the utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in Type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER T2 :Rosa26 tdTomato mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were Type II. Our findings show that most neonatally-derived hair cells become Type II, and many Type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.

Organization of subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) identified with cytoarchitecture and molecular expression.

The auditory system of echolocating bats shows remarkable specialization likely related to analyzing echoes of sonar pulses. However, significant interspecies differences have been observed in the organization of auditory pathways among echolocating bats, and the homology of auditory nuclei with those of non-echolocating species has not been established. Here, in order to establish the homology and specialization of auditory pathways in echolocating bats, the expression of markers for glutamatergic, GABAergic, and glycinergic phenotypes in the subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) was evaluated. In the superior olivary complex, we identified the medial superior olive and superior paraolivary nuclei as expressing glutamatergic and GABAergic phenotypes, respectively, suggesting these nuclei are homologous with those of rodents. In the nuclei of the lateral lemniscus (NLL), the dorsal nucleus was found to be purely GABAergic, the intermediate nucleus was a mixture of glutamatergic and inhibitory neurons, the compact part of the ventral nucleus was purely glycinergic, and the multipolar part of the ventral nucleus expressed both GABA and glycine. In the inferior colliculus (IC), the central nucleus was found to be further subdivided into dorsal and ventral parts according to differences in the density of terminals and the morphology of large GABAergic neurons, suggesting specialization to sonar pulse structure. Medial geniculate virtually lacked GABAergic neurons, suggesting that the organization of the tectothalamic pathway is similar with that of rodents. Taken together, our findings revealed that specialization primarily occurs with regard to nuclei size and organization of the NLL and IC.

Prostaglandins E2 and D2-regulators of host immunity in the model parasite Diphyllobothrium dendriticum: An immunocytochemical and biochemical study.

The spectrum of immunomodulating molecules produced by tapeworms is not yet well understood. The aims of this study, on the tapeworm Diphyllobothrium dendriticum, were: 1) detection and quantification of prostaglandins (PGs) E2 and D2 by high performance liquid chromatography; 2) visualization of PGE2 and PGD2 in specific cells, using methods of immunocytochemistry and confocal laser scanning microscopy; and 3) investigation of the ultrastructure of the cells potentially producing PGE2 and PGD2. The PGE2 immunoreaction (IR) was found in the apical terminals of the frontal glands and sensory organs in the tegument and in small neurons belonging to the main cords and commissures. PGE2-IR partly coincided with α-tubulin-IR. PGD2-IR occurred in the muscle fibers of longitudinal and transverse body muscles and coincided with phalloidin TRITC staining. Both PGE2 and PGD2 were found in the flame cells of the excretory system. Ultrastructural study of the tegument revealed two types of structures that potentially produce PGE2: ciliated and unciliated free nerve endings and frontal gland terminals reinforced with neurotubules. In the main nerve cords, small neurons were identified as potentially exhibiting PGE2-immunoreactivity. In homogenates of the plerocercoids, the measured content of PGE2 and PGD2 was 33.15ngmg-1 and 1.94ngmg-1 of fresh tissue weight, respectively. We found evidence of PGE2 and PGD2 in D. dendriticum parasitizing Coregonus autumnalis (fish) and proved excretion of PGE2 and PGD2 in response to C. autumnalis blood serum. Prostaglandins produced by D. dendriticum probably play a dual role: 1) PGE2 and PGD2 potentially modulate the fish antiparasitic immune response; 2) PGE2 is presumably necessary for proper development and function of the nervous system, and PGD2 can act as an antagonist against mediators causing muscle contraction.

A complex sensory organ in the nose skin of the prosimian primate Lemur catta.

Most mammals have nose tips covered by glabrous skin, a labronasal area, or rhinarium. The surface of the rhinarium of Lemur catta has a dermatoglyphic pattern consisting of epidermal domes. Below the domes, epidermal pegs dip down into the dermis. In and below the tip of the epidermal peg, a complex sensory organ is found. It consists of an association of innervated Merkel cells, lamellate (Pacini-like) bodies with a central nerve, and a ring of unmyelinated nerve endings in the epidermis. The Merkel cells are situated basally in the epidermis and the lamellated bodies just below the epidermis. The unmyelinated nerve endings related to the organ ascend in a circle straight through the epidermis ending below the corneal layer. From these nerve terminals, horizontal spikes enter the keratinocytes. The three components occur together forming an organ and are innervated from a common nerve plexus. The morphology of the complex sensory organ of the lemur shares most crucial components with Eimer's organs in moles, echidna, and platypus, while some structures are lacking, for example, the specific central pillar of keratinocytes, the cuticular cap, and a central unmyelinated fiber. The presence of the essentials of an Eimer's organ in many mammals suggests that a wider definition is motivated.

Afferent nerve ending density in the human laryngeal mucosa: potential implications on endoscopic evaluation of laryngeal sensitivity.

Laryngeal sensitivity is crucial for maintaining safe swallowing, thus avoiding silent aspiration. The sensitivity test, carried out by fiberoptic endoscopic examination of swallowing, plays an important role in the assessment of dysphagic patients. The ventricular folds appear to be more sensitive than the epiglottis during the sensitivity test. Therefore, this study aimed to investigate the mechanical sensitivity of the supraglottic larynx. In seven healthy adults undergoing microlaryngoscopy to remove vocal cord polyps, we excised mucosal samples from the epiglottis and ventricular folds. We measured afferent nerve fiber density by immunoelectron microscopy. All of the subjects underwent an endoscopic sensitivity test based on lightly touching the laryngeal surface of the epiglottis and ventricular folds. The discomfort level was self-rated by the subjects on the visual analog scale. Samples were fixed and stored in cryoprotectant solution at 4 °C. Sections were stained with the protein gene product 9.5, a pan-neuronal selective marker. Nerve fiber density was calculated as the number of fibers per millimeter length of section. The mean nerve fiber density was higher in ventricular samples than in epiglottis samples (2.96 ± 2.05 vs 0.83 ± 0.51; two-sided p = 0.018). The mean visual analog scale scores were significantly higher for touching the ventricular folds than for touching the epiglottis (8.28 ± 1.11 vs 4.14 ± 1.21; two-sided p = 0.017). The higher sensitivity of the ventricular region should be considered for further refining clinical endoscopic evaluation of laryngeal sensitivity.

Identification of different types of spinal afferent nerve endings that encode noxious and innocuous stimuli in the large intestine using a novel anterograde tracing technique.

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Peri-implant bone innervation: histological findings in humans.

PURPOSE: The aim of the present study was to describe nerve fibres around osseointegrated implants in humans. MATERIALS AND METHODS: Twelve mechanically failed implants, retrieved from 10 patients were collected from three dental centres over a period of 5 years. After implant removal, decalcified semi-thin sections (0.5 µm) were stained with thionic methylene blue for light microscopic analysis. In addition, an ultrastructural analysis was performed on serial ultra-thin sections (0.06 µm) using transmission electron microscopy. RESULTS: Both myelinated and unmyelinated nerve fibres could be identified inside the Haversian canals of the osteonal bone near the implant threads. Myelinated fibres were also located at the woven bone around the implant. However, no differentiated nerve endings could be observed around the implants. CONCLUSIONS: This study shows the presence of nerve fibres in human peri-implant bone. Previous studies in animals showed that those fibres participate in the process of bone modelling and remodelling. Yet, the role of peri-implant bone innervation in the osseoperception phenomenon cannot be ruled out since the mechanism of mechanoreception in bone is not fully understood.

In vivo two-photon microscopy of single nerve endings in skin.

Nerve endings in skin are involved in physiological processes such as sensing(1) as well as in pathological processes such as neuropathic pain(2). Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with accumulations of disease specific PrP (PrP(d)) in the central nervous system (CNS) and often the lymphoreticular system (LRS). Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d) in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d) were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d) accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d) from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d) accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d) is at the level of plasma membranes. However, the precise nature of PrP(d)-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d) with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.

Responses of hair follicle-associated structures to loss of planar cell polarity signaling.

The mammalian hair follicle unit consists of a central follicle and a series of associated structures: sebaceous glands, arrector pili muscles, Merkel cells, and sensory nerve endings. The architecture of this multicellular structure is highly polarized with respect to the body axes. Previous work has implicated Frizzled6 (Fz6)-mediated planar cell polarity (PCP) signaling in the initial specification of hair follicle orientation. Here we investigate the origin of polarity information among structures within the hair follicle unit. Merkel cell clusters appear to have direct access to Fz6-based polarity information, and they lose polarity in the absence of Fz6. By contrast, the other follicle-associated structures likely derive some or all of their polarity cues from hair follicles, and as a result, their orientations closely match that of their associated follicle. These experiments reveal the interplay between global and local sources of polarity information for coordinating the spatial arrangement of diverse multicellular structures. They also highlight the utility of mammalian skin as a system for quantitative analyses of biological polarity.

Immunohistochemistry and ultrastructural characteristics of nerve endings in the oral mucosa of rat.

The sensory nerve endings of the rat tongue, cheek and palate were studied using immunohistochemical staining and transmission electron microscopy analysis. The specimens were fixed in modified Karnovsky solution and embedded in Spurr resin. Substance P, calcitonin gene-related peptide (CGRP)- and protein gene product 9.5 (PGP b9.5)-containing nerve fibers in the rat tongue, cheek and palate were examined by electronic microscopical analysis and immunohistochemical localization. These fibers run very close to the basal lamina of the epithelium and extend into the filliform and fungiform papillae. Numerous plexiform fibers immunoreactive for substance P, CGRP and PGP 9.5 were found in the connective tissue of mucosa. Electron microscopic observations showed clearly immunostained nerve fibers, which are located very close to the basal lamina of epithelial cells. Some electron-dense granules may be observed in the axoplasms of both substance P and CGRP immunoreactive fibers. Several lamellar corpuscles into the subepithelial connective tissue papillae, Merkel corpuscles and numerous thin unmyelinated and myelinated axons were observed. The terminal axons revealed numerous mitochondria, neurofilaments, microtubules and clear vesicles in the base of axoplasmic protrusions. The lamellar cells showed caveolae and interlamelar spaces filled by amorphous substance. Between the lamellar cells and axoplasmic membrane, and in the adjacent lamellae region, desmosome-type junctions were observed. The quantitative and morphometric analysis showed nerve endings with an average area of 4.83 ± 3.4 µm(2) and 19.4 internal mitochondria in this site and the organized corpuscles with an average area of 79.24 ± 27.24 µm(2) and 24.23 internal mitochondria in this place. All the structures observed are involved in the transmission of pain and mechanoreceptors stimulus of these oral mucosae.

Glutamatergic neurons in the lateral periaqueductal gray innervate neurokinin-1 receptor-expressing neurons in the ventrolateral medulla of the rat.

The neural pathways underlying the respiratory responses elicited by electrical or chemical stimulation of the lateral part of the periaqueductal gray (lPAG) remain unsettled. In the present study, we examined the lPAG projection to neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the ventrolateral medulla (VLM) which have been implicated in the control of respiration. After biotinylated dextranamine (BDA) injection into the lPAG, NK1R-ir neurons in the rostral VLM were embedded in the plexus of BDA-labeled fibers. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites and additionally with somata of the NK1R-ir neurons. Using retrograde tracing combined with in situ hybridization, we demonstrated that the vast majority of the lPAG neurons projecting to the rostral VLM were positive for vesicular glutamate transporter 2 (VGLUT2) mRNA, but not for glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde tracing and immunohistochemistry, we further demonstrated that the lPAG axon terminals with VGLUT2 immunoreactivity made close apposition with the NK1R-ir neuronal profiles in the rostral VLM. These data suggest that lPAG neurons exert an excitatory influence on NK1R-expressing neurons in the rostral VLM for the control of respiration.