Synaptic morphology of inner and outer hair cells of the human organ of Corti

Innervations of inner and outer hair cells of the organ of Corti of the human cochlea were studied by serial section electron microscopy. At the base of inner hair cells, presumed afferent fibers were of varying size and demonstrated synaptic specialization consisting of a presynaptic body, vesicles, and asymmetrical synaptic membrane specialization. Two types of neurons, vesiculated presumably efferent and nonvesiculated presumably afferent, synapsed at the base of outer hair cells. The synaptic specialization of afferent fibers included presynaptic body, vesicles, and asymmetrical membrane thickening, whereas efferent synapses demonstrated presynaptic vesicles and a subsynaptic cisterna. Some presumably afferent nerve terminals formed a reciprocal synapse with outer hair cells in both the human and the chimpanzee. Such a synaptic relationship demonstrated morphologic specialization consistent with both hair cell-to-neuron and neuron-to-hair cell transmission between the same outer hair cell and nerve terminal. The innervation density of inner and outer hair cells and the comparative anatomy of the afferent and efferent innervation are discussed.     

Glutamatergic innervation in bone

Bone is highly innervated, and evidence for a regulation of bone metabolism by nerve fibers has been suggested by many clinical and experimental studies. However, the nature of the neuromediators involved in these processes has not been well documented. Glutamate (Glu), a major neuromediator of the central nervous system (CNS), was recently identified in nerve fibers running in bone marrow in close contact with bone cells, suggesting that Glu may also act as a neuromediator in this tissue. During the last few years, all the machinery required for glutamate signalling in the CNS was demonstrated in bone. Osteoblasts and osteoclasts express ionotropic Glu receptors (iGluR) (NMDA, AMPA, and Kainate) and metabotropic Glu receptors (mGluR) as well as Glu transporters. Electrophysiological studies have demonstrated that NMDA receptors (NMDAR) and mGluR are functional on bone cells. NMDAR are involved in osteoclast formation and bone resorption and preliminary studies suggest that they may also participate in mechanisms underlying osteoblast proliferation or differentiation, providing evidence for a direct action of Glu on bone cells. The bone loss induced in a model of sciatic neurectomy in growing rats is associated with a decrease of glutamatergic innervation, suggesting that Glu released by nerve fibers may contribute to the regulation of bone remodeling. The manipulation of Glu action in bone may, therefore, represent a new therapeutic target for pathologies associated with modifications of bone remodeling.     

A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.     

Ultrastructure of motor nerve terminals in the anterior third of wistar rat tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957 . For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4°C for 2 h, according to the technique described by Tanaka, 1989 . Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi's apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Neurogenic inflammation in the context of migraine

Despite considerable research into the pathogenesis of idiopathic headaches, such as migraine, the pathophysiological mechanisms underlying them remain poorly understood. Although it is well established that the trigeminal nerve becomes activated during migraine, the consequences of this activation remain controversial. One theory, based on preclinical observations, is that activation of trigeminal sensory fibers leads to a painful neurogenic inflammation within the meningeal (dural) vasculature mediated by neuropeptide release from trigeminal sensory fibres and characterized by plasma protein extravasation, vasodilation, and mast cell degranulation. Effective antimigraine agents such as ergots, triptans, opioids, and valproate inhibit preclinical neurogenic dural extravasation, suggesting that this activity may be a predictor of potential clinical efficacy of novel agents. However, several clinical trials with other agents that inhibit this process preclinically have failed to show efficacy in the acute treatment of migraine in man. Alternatively, it has been proposed that painful neurogenic vasodilation of meningeal blood vessels could be a key component of the inflammatory process during migraine headache. This view is supported by the observation that jugular plasma levels of the potent vasodilator, calcitonin gene-related peptide (CGRP) are elevated during the headache and normalized by successful sumatriptan treatment. Preclinically, activation of trigeminal sensory fibers evokes a CGRP-mediated neurogenic dural vasodilation, which is blocked by dihydroergotamine, triptans, and opioids but unaffected by NK1 receptor antagonists that failed in clinical trials. These observations suggest that CGRP release with associated neurogenic dural vasodilation may be important in the generation of migraine pain, a theory that would ultimately be tested by the clinical testing of a CGRP receptor antagonist.     

High resolution X-ray phase contrast synchrotron imaging of normal and ligation damaged rat sciatic nerves

This study was performed to apply synchrotron radiation (SR) imaging to a neuropathologic evaluation technique after treatment of peripheral nerve blocks. A phase contrast synchrotron images of normal and ligation damaged rat sciatic nerve were obtained with an 8 KeV monochromatic beam and 20-mum thick CsI(TI) scintillation crystal. The visual image was magnified using a 20x microscope objective and captured using an analog CCD camera. Obtained images were compared with conventional light microscopic findings from the same nerve samples. By using an edge enhancement effect of phase const with SR, we could easily discriminate each nerve fiber and identify the arrangement of nerve fibers within a whole thickness (about 1 mm in diameter) of peripheral nerve without sectioning and fixation. The composite SR image of a ligation damaged rat sciatic nerve sample showed that the response to nerve injury was different on each side of the site of injury. The SR image of damaged distal lesion showed destruction of neural microarchitecture and typical extensive Wallerian degeneration of nerve fibers as clearly as histologic image. We could get very detailed morphologic data for Wallerian degeneration of nerve fibers by using the SR imaging technique. We believe that the phase contrast synchrotron imaging has great potential as an imaging tool in the bioscience and medical science.     

Changes in sympathetic nerve activity of the mammalian ovary during a normal estrous cycle and in polycystic ovary syndrome: Studies on norepinephrine release

Although it has been known for many years that the ovary is innervated by catecholaminergic nerve fibers and much experimental evidence has strengthened the notion that catecholamines are physiologically involved in the control of ovarian function, scarce evidence has been presented as to the role of sympathetic activity in ovarian pathologies that affect reproductive function. The purpose of this article is to provide a succinct overview of the findings in this area and discuss them relative to the pathology of polycystic ovary syndrome, the most common ovarian pathology in women during their reproductive years.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Induced maturation of frog mast cells by nerve growth factor during ontogenesis

The effect of nerve growth factor (NGF) on ontogenesis of frog mast cells was investigated in vivo by histochemical, morphometric, and ultrastructural analysis. Three groups of tadpoles at various stages of development were used. In the first group, the larvae received i.p. injections of 1 ng NGF/g; the second group received 10 ng NGF/g, while the control group received only the vehicle. The first recognizable mast cells arose symmetrically in the tongue at stage 26 of Witschi's standard table. At stages 26 and 29, the mast cell number in the NGF-injected tadpoles was significantly higher than the control group. From stage 29 onward, the mast cell number rapidly increased in all groups. No significant differences in mast cell number were observed between the control group and the NGF-injected groups at stages 31 and 33. Electron microscopy revealed that at metamorphic climax (stage 33), the mast cells in the NGF-treated groups were more mature than those in the control group. Therefore, nerve growth factor at early stages of tadpole development is likely to induce differentiation of mast cell precursors, while at later stages it is likely to induce maturation of immature mast cells. The close anatomical association between mast cells and perineurium, observed during nerve development, is intriguing. Already in the early stages of nerve development, the mast cells form a network around Schwann cell-axon complexes, together with the perineurial cells. At climax, the mast cells are located between the perineurial layers, suggesting that they may play a role in the tissue-nerve barrier of the perineurium. Nerve growth factor also seems to induce perineurial cell maturation.     

FIB/SEM characterization of carbon-based fibers

The aim of this paper is to show how a focused ion beam combined with a scanning electron microscope (FIB/SEM machine) can be adopted to characterize composite fibers with different electrical behavior and to gain information about their production and modification. This comparative morphology investigation is carried out on polyacrylonitrile (PAN) carbon fibers and their chemical precursor (the oxidized PAN or oxypan) which has different electrical properties. Fibers are imaged by electron and ion beams and sectioned by the focused ion beam (FIB). A sample of oxypan fibers processed by a radio frequency (RF) plasma is also investigated and the role of the conductive carbon layer around their unmodified, insulating bulk is discussed. A suitable developed edge detection technique (EDT) on electron, ion images, and after the FIB sectioning, provides quantitative information about the thickness of the created layer.     

Localization of calretinin in the rat ovary and in relation to nerve cell bodies in dorsal root and paravertebral ganglia projecting to the ovary

Retrograde tracing with True Blue was combined with immunocytochemistry to determine the source of any calretinin-immunoreactive (CR-ir) nerves projecting to the rat ovary. In the ovary, a strong signal for calretinin immunoreactivity was localized in interstitial gland cells; however, no intraovarian CR-ir nerves could be demonstrated. When the superior ovarian nerve was isolated, cut, and True Blue applied to the proximal end, the fluorescent dye was retrogradely transported to a population of cells located in T-12, T-13, and L-1 dorsal root and paravertebral ganglia. There was virtually no dual labeling of cells in these ganglia with calretinin (< 0.009% dual labeling in dorsal root and <0.014% in paravertebral ganglia). However, greater than two-thirds of the True Blue-labeled cells were immediately adjacent to CR-ir cells in dorsal root ganglia. This arrangement is suggestive of a paracrine mechanism between CR-ir cells and cells projecting to the ovary. In paravertebral ganglia, 63% of cells projecting to the ovary were surrounded completely or partially by beaded CR-ir nerve fibers. The source of these fibers (sensory or preganglionic sympathetic) is unknown but hypothesized to be preganglionic. Collectively, these observations suggest a participatory role for calretinin in ovarian function, either directly via effects on the interstitial gland or indirectly by influencing neurons projecting to the ovary.     

Multiphoton microscopic imaging of human normal and cancerous oesophagus tissue

In this paper, microstructures of human oesophageal submucosa are evaluated using multiphoton microscopy, based on two‐photon excited fluorescence and second harmonic generation. The content and distribution of collagen, elastic fibers and cancer cells in normal and cancerous submucosa layer have been distinctly obtained and briefly discussed. The variation of these components is very relevant to the pathology in oesophagus, especially in early oesophageal cancer. Our results further indicate that the multiphoton microscopy technique has the potential application in vivo in clinical diagnosis and monitoring of early oesophageal cancer.     

Immunoelectron microscopic studies on protein gene product 9.5 and calcitonin gene-related peptide in vallate taste cells and related nerves in the guinea pig

On the basis of our previous report that protein gene product 9.5 (PGP 9.5)-immunoreactive nerve fibers and taste cells and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers are found in guinea pig vallate papillae [Huang and Lu (1996b) Arch. Histol. Cytol. 59:433-441]. We speculated that PGP 9.5 might be a marker for taste receptor cells and that CGRP might play an important role in taste transmission. We, therefore, performed an immunohistochemical and ultrastructural analysis of taste cells and related nerves in guinea pig vallate papillae. In the connective tissue of the vallate papilla, the ultrastructural data revealed that the PGP 9.5-immunoreactive nerve fibers were both myelinated and unmyelinated. The CGRP-immunoreactive nerve fibers were unmyelinated and surrounded by the cytoplasm of Schwann cells as were the non-immunoreactive fibers. In the vallate taste buds, only type III cells, which make synaptic contacts with intragemmal nerves, were PGP 9.5-immunoreactive, while the nerve terminals making synaptic contact with the underlying type III cells were CGRP-immunoreactive. From these observations, we conclude that: (1) PGP 9.5 might be a useful specific marker for type III cells in guinea pig vallate taste buds; and (2) CGRP-containing nerve fibers might be primarily involved in the neural transmission of taste stimuli.     

Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone

Bone alters its metabolic and anabolic activities in response to the variety of systemic and local factors such as hormones and growth factors. Classical observations describing abundance of the nerves fibers in bone also predict a paradigm that the nervous system influences bone metabolism and anabolism. Identification of the nerve-derived signaling molecules, capable of modulating cellular activities of the bone cells, facilitates a novel approach to study the biology of skeletal innervation. Many of the signaling molecules that may act as efferent agents on the bone cells fall into the category of neuropeptides. The present article reviews current understanding of the skeletal innervation and their proposed physiological effects on bone metabolism, with a special interest to calcitonin gene-related peptide (CGRP)-containing nerves fibers. CGRP is abundantly distributed in bone via sensory nerves, especially in the epiphyseal trabecular bones. Its in vitro actions to the cultured osteoblasts and osteoclasts, together with its in vivo localization, strongly support the paradigm that the nervous system influences bone metabolism. In addition, CGRP is recently shown to be expressed endogenously by the osteoblasts. Transgenic mice with osteoblasts overexpressing CGRP are characterized by increased bone formation rate and enhanced bone volume, suggesting that CGRP indeed acts on bone metabolism not only via nervous route but also via autocrine loop. The current article also reviews the distribution of nerve fibers containing substance P (SP), another sensory nerve-specific neuropeptide, and tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine. The distinct effects of SP and catecholamines on the bone cells together with their in vivo influences manifested by experimental denervation studies suggest that the sensory and sympathetic nerves play important roles in bone metabolism.     

Innervation of the carotid body: Immunohistochemical,denervation, and retrograde tracing studies

This review presents information about multiple neurochemical substances in the carotid body. Nerve fibers around blood vessels and glomus cells within the chemoreceptive organ contain immunoreactivities (IR) for tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), calretinin (CR), calbindin D-28k (CB), parvalbumin (PV), and nitric oxide synthase (NOS). Parasympathetic neurons scattered around the carotid body contain VIP, choline acetyltransferase, and vanilloid receptor 1-like receptor. In the mammalian carotid body, transection of the carotid sinus nerve (CSN) causes the absence or decrease of CGRP-, SP-, and NOS-immunoreactive (IR) nerve fibers, whereas all NPY-IR nerve fibers disappear after removal of the superior cervical ganglion. Most VIP-IR nerve fibers disappear but a few persist after sympathetic ganglionectomy. In addition, the CSN transection appears to cause the acquisition of GAL-IR in originally immunonegative glomus cells and nerve fibers within the rat carotid body. On the other hand, 4%, 25%, 17%, and less than 1% of petrosal neurons retrogradely labeled from the rat CSN contain TH-, CGRP-, SP-, and VIP-IR, respectively. In the chicken carotid body, many CGRP- and SP-IR nerve fibers disappear after vagus nerve transection or nodose ganglionectomy. GAL-, NPY-, and VIP-IR nerve fibers mostly disappear after removal of the 14th cervical ganglion of the sympathetic trunk. The origin and functional significance of the various neurochemical substances present in the carotid body is discussed.     

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.     

Nerve fibers innervating the cranial and spinal meninges: morphology of nerve fiber terminals and their structural integration

Pachymeninx and leptomeninx of cranial cavity and spine are considerably different in their collagenous fiber texture, cellular composition, vascularization, and innervation. The majority of meningeal nerve fibers terminate as free nerve endings whereas encapsulated and lamellated nerve terminals additionally occur in higher vertebrates including man. With respect to nerve fiber classification, arborization pattern, topography, and organization of the microenvironment at the termination site afferent and efferent nerve terminals are differentiated. Only the dura mater and the pial subcompartment of the leptomeninx possess the morphological prerequisites for neurogenic inflammation. In the current review, the results of morphological studies regarding the meningeal innervation including the sites of CSF (cerebrospinal fluid) production and absorption are discussed with emphasis on their structure-function relationships.     

Entropy of corneal nerve fibers distribution observed by laser scanning confocal microscopy: A noninvasive quantitative method to characterize the corneal innervation in Sjogren's syndrome patients

Sjogren's syndrome (SS) is a progressive autoimmune condition mainly affecting the salivary and lacrimal glands with an incidence of primary SS between 1/100 and 1/1,000. SS implies an alteration in the epithelium and subepithelium innervation, with consequent reduction of corneal sensitivity. It is necessary to have noninvasive quantitative methods to characterize the status of the corneal nerve fibers of the patients in order to choose and follow the best therapy. Entropy (information dimension) of the nerve corneal fibers distribution observed by confocal microscopy was evaluated in patients with primary SS (n = 30, 6 males, 24 females, 21–81 years), diagnosed by biopsy of salivary gland and blood tests and in sex‐ age‐matched healthy subjects (n = 12). Corneal nerve fiber density, Langerhans cell count, and cell density in the nerve plexus images were also evaluated. In selected patients salivary gland atrophy degree was also evaluated. Nerve corneal distribution observed by confocal microscopy is fractal. Entropy of the corneal nerve distribution statistically distinguishes between SS patients and healthy subjects: patients present a lower value of information dimension of the corneal nerve fibers distribution than healthy individuals (P < 0.001). Percentage of grouped cases classified by entropy according to the subjects (selected patients vs. healthy) showed a 100% sensitivity and 96% specificity, P < 0.0001 with a low value of coefficient of variation among the individuals (6–7 times lower than the other morphometric indexes). Entropy correlated with the severity of the disease (salivary gland atrophy degree, P < 0.01). Evaluation of entropy of the corneal nerve distribution observed by a laser confocal microscopy appears to quantitatively and noninvasively characterize an aspect of the SS patients in relation to the recognition of an impairment of their ocular surface, giving us for the first time a method to objectively and precisely characterize the corneal innervation status in the SS patients. Microsc. Res. Tech. 78:1069–1074, 2015. © 2015 Wiley Periodicals, Inc.     

Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30, and 100 microns solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances. They exhibited a high fluorescence signal at the level of the myelin sheath. Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z-series of stacks of optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship with the afferent fibers.