Nerves in liver: superfluous structures? A special issue of The Anatomical Record updating our views on hepatic innervation.
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Animals↗
Biomedical subjects
Jean-Pierre Timmermans
Publications and source records attributed to Jean-Pierre Timmermans.
Mice lacking Dfna5 show a diverging number of cochlear fourth row outer hair cells.
A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.
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Structure of the respiratory tract of the red-bellied newt Cynops pyrrhogaster, with reference to serotonin-positive neuroepithelial endocrine cells.
To investigate the distribution of serotonin-positive neuroepithelial endocrine (NEE) cells in the respiratory tract of the red-bellied newt Cynops pyrrhogaster, serial sections of the respiratory tract were stained using immunohistochemical methods for serotonin, a periodic acid-Schiff method and hematoxylin-eosin staining. The respiratory tract of the red-bellied newt consisted of a tubular laryngotrachea and two pulmonary sacs. Serotonin-positive solitary NEE cells were distributed widely throughout the ciliomucous epithelium of the laryngotrachea. The innervated clusters of NEE cells, termed neuroepithelial bodies, were rarely found in the caudal portion of the laryngotrachea. The density of NEE cells was high in the middle portion of the laryngotrachea, particularly in the region surrounded by a striated laryngeal sphincter, and the densities were low cranially towards the aditus laryngis and caudally in the pulmonary sacs, the epithelium of which consisted of both ciliomucous epithelial cells and non-ciliated respiratory epithelial cells or pneumocytes. The NEE cells were not observed in the vicinity of non-ciliated respiratory epithelial cells. The morphology and distribution of serotonin-positive NEE cells in the respiratory tract of the red-bellied newt were similar to those described in mammals, including humans. The close localization of NEE cells with ciliary and mucous cells and the distribution of NEE cells in the laryngotrachea suggest that NEE cells play important roles in the defense and repair of the ciliomucous epithelium, such as increasing mucous secretion, ciliary beat frequency and the proliferation of epithelial cells.
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Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43).
The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.
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Immunocytochemical properties of stellate ganglion neurons during early postnatal development.
Neurotransmitter features in sympathetic neurons are subject to change during development. To better understand the neuroplasticity of sympathetic neurons during early postnatal ontogenesis, this study was set up to immunocytochemically investigate the development of the catecholaminergic, cholinergic, and peptidergic phenotypes in the stellate ganglion of mice and rats. The present study was performed on Wistar rats and Swiss mice of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, and 60-day-old). To this end, double labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), vasoactive intestinal (poly)peptide (VIP), neuropeptide Y (NPY), galanin (GAL), and somatostatin (SOM) was applied. The results obtained indicate that the majority of the neurons in the stellate ganglion of both species were TH-positive from birth onward and that a large part of these neurons also contained NPY. The percentage of neurons containing TH and NPY invariably increased with age up to 60 days postnatally. A smaller portion of the stellate ganglion neurons contained other types of neuropeptides and showed a distinct chronological pattern. The proportion of VIP- and ChAT-positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age, whereas the number of SOM-positive cells in rats significantly decreased from birth onward. In newborn rats, VIP-, ChAT- and SOM-positive neurons were largely TH-positive, while their proportions decreased in 10-day-old and older rats. Accordingly, the largest part of VIP-positive neurons also expressed SOM immunoreactivity at birth, after which the number of neurons containing both peptides diminished. The VIP- and SOM-positive cells did not contain NPY in any of the age groups studied. In rats up to 10 days of life, GAL-immunoreactive (-IR) neurons were scarce, after which their number increased to reach a maximal value in 30-day-old animals and then declined again. The SOM-reactive cells had the smallest size in all rats, while the largest neurons were those containing ChAT. In the mouse stellate ganglion, VIP- and ChAT-IR neurons were larger in comparison to NPY- and TH-IR cells. Our study further revealed some species differences: compared to mice the proportion of neurons containing TH and NPY was higher in rats at all ages under study. Furthermore, no GAL-immunostained neurons were found in mice and the number of SOM-positive cells in mice was limited compared to that observed in rats. In conclusion, the development of neurotransmitter composition is complete in rats and mice by their second month of life. At this age, the percentages of immunopositive cells have become similar to those reported in adult animals.
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Coexistence of non-adrenergic non-cholinergic inhibitory and excitatory neurotransmitters in a large neuronal subpopulation in the vaginal segment of the chicken oviduct.
The presence, distribution and smooth muscle motor effects of galanin and pituitary adenylate cyclase activating peptide (PACAP) were studied in the nerves of the vaginal part of the oviduct of egg-laying hens. Galanin and PACAP immunoreactivity were found both in neuronal perikarya and nerve fibres within the wall of the vaginal segment. Both populations showed a similar distribution pattern. Particularly the circular muscle and the intramural vascular net were richly innervated. A few galanin- and PACAP-IR nerve fibres extended up to the mucosal folds. Multiple labelling showed galanin to be colocalised with PACAP as well as with vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) in a large, partly intrinsic neuronal subpopulation innervating the smooth muscle wall. Pharmacological in vitro experiments showed that isolated vaginal muscle strips had a spontaneous basal activity that was not affected by the neuronal conductance blocker tetrodotoxin (TTX). Galanin induced concentration-dependent contractions that were TTX-insensitive. PACAP, VIP, nitric oxide (NO) and the NO donor nitroglycerin caused concentration-dependent relaxations that were TTX-insensitive. Electrical field stimulation of isolated muscle strips induced frequency-dependent relaxations that were blocked by TTX and reduced by the NOS blocker L-nitroarginine. These data provide evidence that the vaginal part of the oviduct contains a largely intrinsic, neuronal subpopulation, capable of releasing multiple non-adrenergic, non-cholinergic (NANC) motor agents for the control of local muscular activities. In addition, we provided pharmacological evidence that VIP, NO and PACAP exert an inhibitory and galanin an excitatory action on isolated muscle strips of the vaginal part of the chicken oviduct. Our results suggest that these NANC neurotransmitters play an important role in the regulation of neuromuscular activity in this region.
Adrenergic Agents↗
Further characterisation of particulate neuronal nitric oxide synthase in rat small intestine.
Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.
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Circling behavior in the Ecl mouse is caused by lateral semicircular canal defects.
The epistatic circler mouse (Ecl mouse) is a preexisting mutant, which displays a circling phenotype and hyperactivity. It has been shown that the circling phenotype in this mutant results from a complex inheritance pattern, but the vestibular pathology has not been analyzed. The present study deals with the morphological and functional basis responsible for the circling behavior in the Ecl mouse. Morphological examination of the inner ears revealed a bilateral malformation of the horizontal (lateral) semicircular canal and duct. No cochlear abnormalities were detected, and auditory brainstem response (ABR) measurements indicated that the auditory system is not affected. Investigation of the vestibuloocular reflex (VOR) in Ecl mice showed that their horizontal VOR on stimulation is virtually absent, which correlates with the morphological findings.
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Neuropeptide-containing neurons in the endopiriform region of the rat: morphology and colocalization with calcium-binding proteins and nitric oxide synthase.
The endopiriform nucleus, further divided into dorsal and ventral parts, and the neighbouring pre-endopiriform (pEn) nucleus form a region of highly heterogeneous structure involved in numerous physiological and pathological processes. Nonpyramidal neurons of this region containing three neuropeptides-somatostatin (SOM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP)-were examined in this study. Their colocalization with three calcium-binding proteins-parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and with nitric oxide synthase (NOS), was investigated by qualitative and quantitative methods. The results are summarized as follows: (1) all studied substances are distributed in neurons of the entire region, (2) SOM-ir neurons constitute the most numerous neuropeptide-containing population, whereas NOS-ir neurons make up the largest population of all studied, (3) colocalizations are found in the endopiriform region (Enr) (SOM with CB, PV and NOS; VIP with CR; NPY with NOS and NOS with CR), (4) heterogeneity of the endopiriform region appears in the differences of cells' shape distributions of single-labeled (SOM-, CR-PV-ir) and double-labeled (SOM/CB-, SOM/PV-, NPY/NOS- and NOS/CR-ir) neurons, as well as in differentiated percentage values of SOM/NOS, NPY/NOS and VIP/CR double-labeled neurons in three studied parts; additionally, differences in distribution of immunoreactive neuropil elements between parts of the region are observed. Numerous regional differences concerning neuronal morphology and immunocytochemical characteristics justify further division of the endopiriform region into distinguished parts. Some immunocytochemical features of the neurons in studied region may contribute to the role in epileptogenesis.
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Purinergic signalling in the lung: important in asthma and COPD?
During the past decade, evidence has accumulated for the involvement of purines and purine receptors in the pathogenesis and pathophysiology of asthma and chronic obstructive pulmonary diseases. In particular, the indirect bronchospasmic and inflammatory actions of purines via the activation and degranulation of mast cells has been extensively studied and reviewed. At present, interest is focused on the impact of purines on pulmonary innervation and the neuroendocrine system, and the potent interaction of the latter with many of the airway components that are essentially altered in asthma and chronic obstructive pulmonary disease. However, data related to these interactions are diverse and not always easily recognised in the literature.
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Segmental distribution and morphometric features of primary sensory neurons projecting to the tibial periosteum in the rat.
Previous reports have demonstrated very rich innervation pattern in the periosteum. Most of the periosteal fibers were found to be sensory in nature. The aim of this study was to identify the primary sensory neurons that innervate the tibial periosteum in the adult rat and to describe the morphometric features of their perikarya. To this end, an axonal fluorescent carbocyanine tracer, DiI, was injected into the periosteum on the medial surface of the tibia. The perikarya of the sensory fibers were traced back in the dorsal root ganglia (DRG) L1-L6 by means of fluorescent microscopy on cryosections. DiI-containing neurons were counted in each section and their segmental distribution was determined. Using PC-assisted image analysis system, the size and shape of the traced perikarya were analyzed. DiI-labeled sensory neurons innervating the periosteum of the tibia were located in the DRG ipsilateral to the injection site, with the highest distribution in L3 and L4 (57% and 23%, respectively). The majority of the traced neurons were of small size (area < 850 microm2), which is consistent with the size distribution of CGRP- and SP-containing cells, regarded as primary sensory neurons responsible for perception of pain and temperature. A small proportion of labeled cells had large perikarya and probably supplied corpuscular sense receptors observed in the periosteum. No differences were found in the shape distribution of neurons belonging to different size classes.
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Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies.
Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.
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Quantification of neuroepithelial bodies and their innervation in fawn-hooded and Wistar rat lungs.
The Fawn-Hooded rat (FHR), a model for primary pulmonary hypertension, shows an unexplained hypersensitivity to airway hypoxia. Because pulmonary neuroepithelial bodies (NEBs) appear to express a functional oxygen-sensing mechanism and an extensive sensory innervation, possible changes in this system should be taken into consideration. In the present study a comparative analysis of NEBs and their selective innervation was performed in FHRs and Wistar control rats. In both rat strains, the number of NEBs was estimated to be around 3,500, approximately 40% of which were innervated by vagal sensory calbindin D28k-immunoreactive (IR) nerve endings and approximately 50% by spinal sensory calcitonin gene-related peptide (CGRP)-IR nerve terminals. The number of intrinsic pulmonary nitrergic neurons and the percentage of pulmonary NEBs revealing a nitrergic innervation were highly significantly lower in FHRs. In both FHRs and Wistar rats, a remarkable morphologic interaction was observed between the intrinsic nitrergic and the CGRP-IR sensory population contacting NEBs. Our findings suggest a possible link between the hypersensitivity to airway hypoxia observed in FHRs and a reduced intrinsic pulmonary nitrergic innervation, possibly via the interaction with pulmonary NEBs and their spinal sensory CGRP-IR innervation.
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A globin in the nucleus!
Cytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globin-folded proteins as transcriptional regulators.
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Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of Schistosoma mansoni-infected mice.
In the present study, the effect of intestinal schistosomiasis on the extrinsic sensory innervation of the murine ileum was investigated. Immunocytochemical techniques to localize calcitonin gene-related peptide (CGRP), substance P (SP), and vanilloid receptor 1 (VR1) were combined with retrograde tracing techniques and capsaicin treatment. Neurochemical characterization of extrinsic primary afferent neurons (EPANs) in normal and capsaicin-treated mice, revealed that CGRP and VR1, but not SP, were expressed in extrinsic afferents. Immunocytochemical analysis using the above-mentioned antibodies yielded three different populations of neurons in both dorsal root and nodose ganglia, namely CGRP/--, SP/--, and CGRP/SP-expressing neurons. Retrograde tracing revealed that only CGRP/--expressing neurons projected to the ileum. Intestinal schistosomiasis resulted in an upregulation of the number of CGRP-immunoreactive (ir) nerve fibers in the lamina propria of the villi, coinciding with an increase in mucosal mast cells in acutely and chronically infected animals. In infected animals, mucosal mast cells were found closely associated with a dense mucosal CGRP-ir fiber network. Neonatal capsaicin treatment led to a 70% reduction in the number of mucosal mast cells. In conclusion, the present study provides evidence that CGRP is a valid marker for EPANs in the mouse ileum, which are involved in the recruitment of mucosal mast cells. Morphological evidence is provided of a neuroimmune interaction between mucosal mast cells and EPANs in schistosoma-infected mice.
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Myenteric nitrergic neurons along the rat esophagus: evidence for regional and strain differences in age-related changes.
Several studies have suggested an age-related reduction in the number of myenteric neurons in the lower gastrointestinal (GI) tract linked to changes in GI neuromuscular functions. The present study, combining protein gene product 9.5 immunostaining and NADPH-diaphorase histochemistry, aimed at quantifying the proportion of nitrergic neurons compared to the overall number of enteric neurons in the esophagus of young (3-4.5 months) and aged (18-20 months) Sprague-Dawley and Wistar rats. In both strains, the neuron numbers per ganglion in the cervical region were almost twice as high as in the other esophageal regions. Irrespective of age or strain, the esophagus harbored a very high proportion of intrinsic nitrergic neurons (greater than approximately 65%). Both strains showed with aging an overall neuronal loss of approximately 27%. While a significant increase (young: 64-71%; aged: 82-89%) was observed in all esophageal regions in the Wistar strain, the proportion of nitrergic neurons remained stable with aging in the Sprague-Dawley strain (range: 72-82%). In conclusion, the age-related reduction in the overall number of myenteric, nitrergic, and non-nitrergic neurons observed in the rat esophagus, appears to be highly region- and strain-dependent. Therefore, a protective mechanism against neuronal cell loss, selectively present in specific (nitrergic) enteric subpopulations, as suggested in earlier reports, cannot be put forward as a general phenomenon throughout the entire GI tract.
Aging↗
Glial cells, but not interstitial cells, express P2X7, an ionotropic purinergic receptor, in rat gastrointestinal musculature.
Purinergic (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal tract. So far, seven ionotropic purinergic receptors (P2X1-7) have been cloned. As specific antibodies become available, their respective distribution in the gastrointestinal tract can be elucidated. Here, we used high-resolution tricolor confocal microscopy, to study the distribution of P2X7-immunoreactive (-ir) cells in the muscularis propria of the rat stomach, small intestine, and colon. Smooth muscle cells, KIT-ir interstitial cells of Cajal, and CD34/SK3-ir fibroblastlike cells were P2X7-negative, whereas P2X7 immunoreactivity was observed in nerves and S100-ir glial cells. In all regions studied, P2X7 immunoreactivity was also observed in myenteric and submucosal ganglia, where perineuronal nerve endings appeared brightly labeled. Our observations suggest that purinergic signaling could influence the enteric glia through P2X7 receptors.
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The WFS1 gene, responsible for low frequency sensorineural hearing loss and Wolfram syndrome, is expressed in a variety of inner ear cells.
Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.
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