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

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