Single cell analysis of functional and transcriptional changes in somatosensory neurons after peripheral nerve injury

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Project Summary/Abstract Neuropathic pain is associated with the spontaneous activity and hyperexcitability of a small subset of somatosensory neurons. Changes in somatosensory neuron gene expression also follow nerve injury in animal models of neuropathic pain. To date, gene expression studies of somatosensory neurons have analyzed changes in bulk populations of cells. However, bulk studies mask the functional and transcriptional heterogeneity of somatosensory neurons. Thus, it is still unclear which subpopulation(s) of somatosensory neurons are the primary drivers of neuropathic pain, nor is it known what transcriptional changes lead to ectopic activity of individual somatosensory neurons after nerve injury. A detailed single cell characterization of electrophysiological and transcriptional changes in somatosensory neurons following nerve injury will greatly expand our understanding of chronic pain and may identify novel drug targets for the treatment of chronic and neuropathic pain. I propose to use calcium imaging in dorsal root ganglion (DRG) neurons from transgenic mice expressing the genetically encoded calcium indicator (GCaMP6f) in vitro to identify ectopically active neurons after nerve injury. After identifying spontaneously active neurons, I will perform electrophysiological recordings on these neurons to confirm the presence of ectopic activity. I will also compare the general excitability of injured and uninjured DRG neurons, and measure their sodium and potassium currents to determine whether nerve injury indeed makes somatosensory neurons hyperexcitable. After characterizing the electrophysiological excitability of individual DRG neurons, I will harvest each cell separately, isolate its mRNA and perform massively parallel single cell qPCR to measure the expression of all known ion channels and neurotransmitter receptors. In addition, I will use transcriptional markers to classify individual DRG neurons into separate subtypes. After obtaining this dataset, I will identify changes in ion channel expression associated with neuronal hyperexcitability after injury and determine in which subtype(s) of sensory neurons these changes occur. This analysis should help identify the cellular and molecular drivers of injury-induced neuropathic pain in the peripheral nervous system.