Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both ...necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.
The spinal cord dorsal horn occupies a key position in the central nervous system, as it is the first site of integration of somatosensory input from the periphery. Local interneurons of the dorsal ...horn process the incoming peripheral information while projection neurons relay it to supraspinal regions via ascending pathways. However, the exact mechanisms through which this occurs in normal or maladaptive states, and the neuron populations involved, remains to be understood. Identification of the neuronal cell types present in the dorsal horn will enable us to determine their functional organization. Thus, a goal of this thesis is to achieve a consensus in the cell types and their classification.To do this, we performed single-nucleus RNA-sequencing (snRNA-seq) of the Rhesus macaque dorsal horn. Mapping out the cellular and molecular organization of the non-human primate allows us to develop a reliable species-independent classification scheme for the cell types that can then facilitate translation of rodent dorsal horn studies to primate. From the snRNA-seq, we identified 11 excitatory neuronal clusters (GLUTs) and 5 inhibitory clusters (GABAs). Based on the cytoarchitecture of the spinal cord, layers or laminae have been described along the dorso-ventral axis, and these appear to correspond well with the functional organization of neuron populations. In situ hybridization revealed that a majority of the clusters reside within specific lamina(e). A comparative analysis between clusters of the macaque and mouse meta-analysis showed a strong correspondence between both species as well as several interesting species-specific differences at the gene level.An additional goal of this thesis is to describe a strategy for the identification of regulatory elements in transcriptionally active regions of individual dorsal horn cell types using single nucleus Assay for Transposase-Accessible Chromatin coupled with sequencing (snATAC-seq). We found that the snATAC-seq derived clusters correspond well with our snRNA-seq clusters i.e., all 16 cell-types identified from the snRNA-seq have good representation in the scATAC-seq data. These data further validate our proposed classification scheme of dorsal horn cell types while also providing candidate regulatory regions that drive expression in specific cell types.
PDF
