Autonomic fiber sprouting in the skin in chronic inflammation

Pain is a major symptom associated with chronic inflammation. In previous work from our laboratory, we have shown that in animal models of neuropathic pain there is a sprouting of sympathetic fibers into the upper dermis, a territory normally devoid of them. However, it is not known whether such sympathetic spouting, which is likely trophic factor mediated, also occurs in chronic inflammation and arthritis. In the present study, we used a rat model of chronic inflammation in which a small single dose of complete Freund's adjuvant (CFA) was injected subcutaneously, unilaterally, into the plantar surface of the hindpaw. This led to a localized long-term skin inflammation and arthritis in all joints of the hindpaw. Animals were perfused with histological fixatives at 1, 2, 3 or 4 weeks after the injection. Experimental animals treated with CFA were compared to saline-injected animals. We then investigated the changes in the pattern of peripheral innervation of the peptidergic nociceptors and sympathetic fibers in rat glabrous hindpaw skin. Antibodies directed towards calcitonin gene-related peptide (CGRP) and dopamine beta-hydroxylase (DBH) were used for the staining of peptidergic and sympathetic fibers, respectively. Immunofluorescence was then used to analyze the different nerve fiber populations of the upper dermis. At 4 weeks following CFA treatment, DBH-immunoreactive (IR) fibers were found to sprout into the upper dermis, in a pattern similar to the one we had observed in animals with a chronic constriction injury of the sciatic nerve in a previous publication. There was also a significant increase in the density of CGRP-IR fibers in the upper dermis in CFA treated animals at 2, 3 and 4 weeks post-injection. The increased peptidergic fiber innervation and the ectopic autonomic fibers found in the upper dermis may have a role in the pain-related behavior displayed by these animals

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn

Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia

Neuronal circuitry for pain processing in the dorsal horn

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region

Changes in the spinal cord and peripheral innervation in an animal model of arthritis

Neurons in the marginal layer (lamina I) of the dorsal horn of the spinal cord play a major role in the transmission and integration of pain-related sensory information that is relayed to the brain. Alteration in excitability of these cells greatly influences pain perception. Among these, alterations of the substance P (SP) system play a major role because of its known involvement in spinal cord nociceptive mechanisms. In this thesis, our main focus was on the characterization of the cell populations in lamina I of the spinal cord which express the SP receptor (NK-1r) and project to the parabrachial nucleus in normal rats and in an animal model of localized polyarthritis. Lamina I projection neurons can be classified into three morphological types, the fusiform, the multipolar (flattened) and the pyramidal. Our combined tract-tracing and immunocytochemical studies demonstrated that in control animals the fusiform and multipolar neurons project abundantly to the parabrachial nuclei and express the NK-1r, whereas pyramidal neurons, although projecting in almost identical proportion to the same target, express little, if any, NK-1r. In rats treated with a single, unilateral low dose subcutaneous injection of complete Freund's adjuvant (CFA) in the thick skin of the hind paw, we demonstrated an ipsilateral de novo NK-1r expression on the normally non-nociceptive spinoparabrachial lamina I pyramidal cell type starting at 15 days post-injection. We also observed, as from 15 days post-CFA, an innervation of pyramidal neurons by SP-immunoreactive (IR) boutons. It should be pointed out that pyramidal neurons are normally not innervated by SP, which would confirm their non-nocicDans la moelle épinière, les neurones de la couche marginale (couche 1) de la corne dorsale jouent un rôle majeur dans l'intégration et la transmission de l'information sensorielle vers le cerveau. Des changements au niveau de l'excitabilité de ces cellules influencent grandement la perception de la douleur. Parmi ceux-ci, un changement au niveau du système de la substance P (SP) peut avoir un grand impact en raison de son implication dans les mécanismes nociceptifs. Dans cette thèse, notre but premier était de caractériser la population neuronale de la couche 1 qui exprime le récepteur de la substance P (NK-1 et qui projette au noyau parabrachial. Les observations se sont faites chez les rats normaux et dans un modèle animal de polyarthrite locale. Les cellules de projection de la couche 1 peuvent être classifiées en trois types morphologiques; les fusiformes, les multipolaires (aplaties) et les pyramidales. Notre approche combinant le traçage rétrograde et l'immunocytochimie a démontré que, chez les animaux contrôles, les neurones fusiformes, multipolaires et pyramidaux projettent abondamment au noyau parabrachial. Les deux premiers types de cellules expriment le récepteur NK-1, alors que les cellules pyramidales l'expriment très peu, sinon pas. Chez les rats traités avec une seule dose unilatérale d'adjuvant complet de Freund (ACF), administrée sous-cutanée dans la plante de la patte, nous avons démontré une nouvelle expression du récepteur NK-1. Les cellules pyramidales qui sont habituellement non nociceptives sont imunoréactives pour le récepteur NK-1 à partir de 15 jours après l'injection. Nous avons aussi observé, à 15 jours ap

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABAB receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 μm2) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy