Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia.

Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.

Tiling and somatotopic alignment of mammalian low-threshold mechanoreceptors.

Innocuous mechanical stimuli acting on the skin are detected by sensory neurons, known as low-threshold mechanoreceptors (LTMRs). LTMRs are classified based on their response properties, action potential conduction velocity, rate of adaptation to static indentation of the skin, and terminal anatomy. Here, we report organizational properties of the cutaneous and central axonal projections of the five principal hairy skin LTMR subtypes. We find that axons of neurons within a particular LTMR class are largely nonoverlapping with respect to their cutaneous end organs (e.g., hair follicles), with Aß rapidly adapting-LTMRs being the sole exception. Individual neurons of each LTMR class are mostly nonoverlapping with respect to their associated hair follicles, with the notable exception of C-LTMRs, which exhibit multiple branches that redundantly innervate individual hair follicles. In the spinal cord, LTMR central projections exhibit rostrocaudal elongation and mediolateral compression, compared with their cutaneous innervation patterns, and these central projections also exhibit a fine degree of homotypic topographic adjacency. These findings thus reveal homotypic tiling of LTMR subtype axonal projections in hairy skin and a remarkable degree of spatial precision of spinal cord axonal termination patterns, suggesting a somatotopically precise tactile encoding capability of the mechanosensory dorsal horn.

The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.

The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.

Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn.

The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states.

Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.