Biomedical subjects

Inge Brouns

Publications and source records attributed to Inge Brouns.

14 recordsLinked to original sources

PubMed2025

Functional synapses between neurons and small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, frequent early relapse and a high mortality rate1-3. Recent evidence has suggested that innervation has an important role in the development and progression of several types of cancer4,5. Cancer-to-neuron synapses have been reported in gliomas6,7, but whether peripheral tumours can form such structures is unknown. Here we show that SCLC cells can form functional synapses and receive synaptic transmission. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified neuronal, synaptic and glutamatergic signalling gene sets in mouse and human SCLC. Further experiments revealed the ability of SCLC cells to form synaptic structures with neurons in vitro and in vivo. Electrophysiology and optogenetic experiments confirmed that cancer cells can receive NMDA receptor- and GABAA receptor-mediated synaptic inputs. Fitting with a potential oncogenic role of neuron-SCLC interactions, we showed that SCLC cells derive a proliferation advantage when co-cultured with vagal sensory or cortical neurons. Moreover, inhibition of glutamate signalling had therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells seem to hijack synaptic signalling to promote tumour growth, thereby exposing a new route for therapeutic intervention.

Animals↗

PubMed2006

Sensory receptors in the visceral pleura: neurochemical coding and live staining in whole mounts.

Today, diagnosis and treatment of chest pain related to pathologic changes in the visceral pleura are often difficult. Data in the literature on the sensory innervation of the visceral pleura are sparse. The present study aimed at identifying sensory end-organs in the visceral pleura, and at obtaining more information about neurochemical coding. The immunocytochemcial data are mainly based on whole mounts of the visceral pleura of control and vagally denervated rats. It was shown that innervation of the rat visceral pleura is characterized by nerve bundles that enter in the hilus region and gradually split into slender bundles with a few nerve fibers. Separate nerve fibers regularly give rise to characteristic laminar terminals. Because of their unique association with the elastic fibers of the visceral pleura, we decided to refer to them as "visceral pleura receptors" (VPRs). Cryostat sections of rat lungs confirmed a predominant location on mediastinal and interlobar lung surfaces. VPRs can specifically be visualized by protein gene product 9.5 immunostaining, and were shown to express vesicular glutamate transporters, calbindin D28K, Na+/K+-ATPase, and P2X3 ATP-receptors. The sensory nerve fibers giving rise to VPRs appeared to be myelinated and to have a spinal origin. Because several of the investigated proteins have been reported as markers for sensory terminals in other organs, the present study revealed that VPRs display the neurochemical characteristics of mechanosensory and/or nociceptive terminals. The development of a live staining method, using AM1-43, showed that VPRs can be visualized in living tissue, offering an interesting model for future physiologic studies.

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PubMed2006

Evidence for a role of neuroepithelial bodies as complex airway sensors: comparison with smooth muscle-associated airway receptors.

The epithelium of intrapulmonary airways in many species harbors diffusely spread innervated groups of neuroendocrine cells, called neuroepithelial bodies (NEBs). Data on the location, morphology, and chemical coding of NEBs in mammalian lungs are abundant, but none of the proposed functions has so far been fully established. Besides C-fiber afferents, slowly adapting stretch receptors, and rapidly adapting stretch receptors, recent reviews have added NEBs to the list of presumed sensory receptors in intrapulmonary airways. Physiologically, the innervation of NEBs, however, remains enigmatic. This short overview summarizes our present understanding of the chemical coding and exact location of the receptor end organs of myelinated vagal airway afferents in intrapulmonary airways. The profuse populations that selectively contact complex pulmonary NEB receptors are compared with the much smaller group of smooth muscle-associated airway receptors. The main objective of our contribution was to stimulate the idea that the different populations of myelinated vagal afferents that selectively innervate intraepithelial pulmonary NEBs may represent subpopulations of the extensive group of known electrophysiologically characterized myelinated vagal airway receptors. Future efforts should be directed toward finding out which airway receptor groups are selectively coupled to the complex NEB receptors.

Animals↗

PubMed2006

Sensory receptors in the airways: neurochemical coding of smooth muscle-associated airway receptors and pulmonary neuroepithelial body innervation.

Mainly due to the lack of conclusive morphological data, correlation between functionally and morphologically defined lung receptors has so far been unsatisfactory. In the present study, multiple immunocytochemical stainings with a panel of markers for (mechanso)sensory nerve fibres were performed in order to visualise putative receptor terminals in rat intrapulmonary airways. We first focussed on determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. Immunostaining with antibodies against Na+/K+-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 revealed branching laminar subepithelial receptor endings associated with airway smooth muscle. The latter nerve terminals appeared to further express calbindin D28k (CB), and the ATP receptor P2X3, but were calcitonin gene-related peptide (CGRP)-negative. The nerve fibres that give rise to these terminals were shown to be myelinated and have a vagal sensory origin. Because of the close association between the laminar terminals of this receptor-like structures and airway smooth muscle, we will further refer to these clearly morphologically identifiable sensory end organs as 'smooth muscle-associated airway receptors (SMARs)'. Secondly, we further explored the sensory innervation of pulmonary neuroepithelial bodies (NEBs). NEBs are intraepithelial groups of neuroendocrine cells, contacted by several nerve fibre populations, at least three of which are sensory. The spinal sensory innervation of NEBs expresses CGRP and substance P, contacts NEBs at their basal pole, and is capsaicin-sensitive. The intraepithelial vagal sensory innervation of NEBs, on the other hand, appears to be myelinated and could be labelled by antibodies against VGLUT1, VGLUT2, CB and P2X3 receptors. Na+/K+-ATPase alpha3 immunostaining additionally labelled part of the vagal sensory innervation of rat pulmonary NEBs. The neurochemical coding and receptor-like appearance of SMARs and of the complex vagal sensory innervation of NEBs appeared to be almost identical and reminiscent of mechanosensors. Both SMARs and vagal nodose nerve terminals in NEBs therefore likely represent the morphological counterparts of subgroups of the extensive population of physiologically characterised myelinated vagal airway receptors, the majority of which are mechanosensitive. Electrophysiological data based on 'local' stimuli should be interpreted with caution, because of the regular close apposition of SMARs and NEBs and the very similar characteristics of their nerve terminals.

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PubMed2005

Neurochemical characterisation of sensory receptors in airway smooth muscle: comparison with pulmonary neuroepithelial bodies.

Descriptions of morphologically well-defined sensory airway receptors are sparse, in contrast to the multiplicity of airway receptors that have been identified electrophysiologically. The present study aimed at further determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. The results were compared with those obtained for pulmonary neuroepithelial bodies (NEBs), which are complex intraepithelial sensory airway receptors. Multiple immunocytochemical staining showed branching laminar subepithelial receptor-like endings, which were found to intercalate in the smooth muscle layer of intrapulmonary conducting airways in rats. Because of the consistent intimate association with the airway smooth muscle, the laminar terminals will further be referred to as 'smooth muscle-associated airway receptors (SMARs)'. SMARs were characterised by their Na(+)/K(+)-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2-immunoreactivity, expression of the ATP receptor P2X(3), and the presence of calcium-binding proteins. Nerve fibres giving rise to SMARs were shown to be myelinated and to have a vagal origin. Interestingly, the neurochemical coding and receptor-like appearance of SMARs appeared to be almost identical to at least part of the complex vagal sensory terminals in NEBs. Intraepithelial nerve endings in pulmonary NEBs were indeed also shown to originate from myelinated vagal afferent nerve fibres, and to express Na(+)/K(+)-ATPase alpha3, VGLUT1, VGLUT2, P2X(3) and calcium-binding proteins. Since several of the latter proteins have been reported as markers for mechanoreceptor terminals in other organs, both SMARs and the vagal nodose nerve terminals in NEBs seem good candidates to represent the morphological counterparts of at least subsets of the extensive population of physiologically characterised myelinated vagal airway mechanoreceptors. The observation that SMARs and NEBs are regularly found in each other's immediate neighbourhood, and the very similar characteristics of their nerve terminals, point out that the interpretation of electrophysiological data based on 'local' stimuli should be made with great caution.

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PubMed2003

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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PubMed2003

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.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors.

Innervated groups of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium of intrapulmonary airways in many species. Our present understanding of the morphology of NEBs in mammalian lungs is comprehensive, but none of the proposed functional hypotheses have been proven conclusively. In recent reviews on airway innervation, NEBs have been added to the list of presumed physiological lung receptors. Microscopic data on the innervation of NEBs, however, have given rise to conflicting interpretations. Using neuronal tracing, denervation, and immunostaining, we recently demonstrated that the innervation of NEBs is much more complex than the almost unique vagal nodose sensory innervation suggested by other authors. The aim of the present work is to summarize our present understanding about the origin and chemical coding of the profuse nerve terminals that selectively contact pulmonary NEBs. A thorough knowledge of the complex interactions between the neuroendocrine cells and at least five different nerve fiber populations is essential for defining the position(s) of NEBs among the many pulmonary receptors characterized by lung physiologists.

Animals↗

PubMed2003

Reduced number of intrinsic pulmonary nitrergic neurons in Fawn-Hooded rats as compared to control rat strains.

The Fawn-Hooded rat (FHR) strain reveals a congenital predisposition to primary (idiopathic) pulmonary hypertension (PPH), and can therefore be regarded as an animal model in which to study possible mechanisms underlying an inherited susceptibility to pulmonary hypertension. Pulmonary hypertension can be induced in FHRs after a short exposure to mild hypoxia, presumably because of an altered peripheral oxygen sensitivity. Given the presence of pulmonary nitrergic neurons in rat lungs, the observed link between airway hypoxia and the expression of pulmonary neuronal nitric oxide synthase (nNOS), and the fact that nNOS appears to be involved in peripheral chemoreceptor sensitivity, we examined the intrinsic pulmonary nitrergic innervation in the FHR. In the present study the number of intrapulmonary nitrergic nerve cell bodies, detected by NADPH diaphorase (NADPHd) histochemistry, was quantified in the FHR and three control rat strains. Compared to the control rat strains, the FHR lungs revealed a highly significantly lower number of intrinsic nitrergic neurons, while no apparent differences were found in the number of enteric nitrergic neurons in the esophagus. In conclusion, the possible links between neuronal NO, hypersensitivity to airway hypoxia, and the development of PPH clearly deserve further investigation.

Animals↗

PubMed2003

Dual sensory innervation of pulmonary neuroepithelial bodies.

The characteristics of the different populations of sensory nerve terminals that selectively contact pulmonary neuroepithelial bodies (NEBs) in rat lungs were investigated after chemical denervation with capsaicin and compared with control lungs. Vagal calbindin D28k and P2X(3) purinoceptor immunoreactive (IR) afferent nerve terminals contacting NEBs appeared to have their origin in the nodose ganglion. Thick CB/P2X(3)-IR nerve fibers were seen to be myelinated and to lose their myelin sheaths just before branching and protruding intraepithelially between the NEB cells. This vagal sensory component of the innervation of NEBs was not affected by capsaicin nor expressed capsaicin receptors (vanilloid receptor subtype 1). A second sensory nerve fiber population that selectively innervates pulmonary NEBs in the rat lung consists of thin unmyelinated nonvagal substance P/calcitonin gene-related peptide IR nerve fibers, contacting mainly the basal pole of pulmonary NEBs, and having their origin in dorsal root ganglia. In concordance with vanilloid receptor 1 expression on these nerve terminals, the spinal sensory substance P/calcitionin gene-related peptide-IR component of the innervation of NEBs was depleted by systemic capsaicin treatment. The complex sensory innervation pattern of pulmonary NEBs characterized in the present study strongly suggests that, physiologically, pulmonary NEBs represent a group of intraepithelial receptors that may be able to accommodate various local and central reflex actions, in relation to both chemo- and mechanosensory stimuli.

Animals↗

PubMed2002

Neuroepithelial bodies: a morphologic substrate for the link between neuronal nitric oxide and sensitivity to airway hypoxia?

Currently, the significance of nitric oxide (NO) in the respiratory tract is a matter of great interest because NO is believed to play a major role in the physiological regulation of airway function but also in lung pathology. What is especially intriguing with respect to the present investigation, are reports that the pulmonary expression of neuronal NO synthase (nNOS) is altered as a result of airway hypoxia. We examined the possible relationship between intrapulmonary nitrergic structures and pulmonary neuroepithelial bodies (NEBs), chemoreceptor-like epithelial cell groups that are known to have all necessary components for oxygen perception. Tyramide-enhanced immunostaining for nNOS was combined with known markers for NEBs in an ontogenetic study of rat lungs. From postnatal day 2 onward, nNOS-immunoreactive (-IR) neuronal cell bodies, present mainly in the lamina propria at all levels of intrapulmonary airways, were seen to give rise to remarkable intraepithelial terminal arborizations that invariably colocalized with NEBs. nNOS immunoreactivity was absent from the vagal calbindin D28k(CB) -IR and the spinal calcitonin gene-related peptide(CGRP) -IR extrinsic sensory nerve fiber populations that our group reported earlier to selectively contact NEBs. Quantitative analysis showed that all NEBs receiving nNOS-IR terminals were also contacted by CGRP-IR nerve fibers, whereas approximately 55% were additionally contacted by CB-IR nerves. The reported nitrergic neurons did not express the cholinergic marker vesicular acetylcholine transporter and were always surrounded by a basket of CGRP-IR nerve terminals. In conclusion, part of the pulmonary NEBs selectively receive extensive nitrergic nerve terminals that originate from intrinsic neurons. Together with literature data on lung physiology and pharmacology, some interesting suggestions for the functional significance of the association between pulmonary CGRP-IR NEBs, nNOS-IR neurons, and CGRP-IR afferents described in the present study, are discussed.

Airway Obstruction↗

PubMed2002

Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum.

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.

Animals↗

PubMed2002

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.

Animals↗