Altered Sensibility Following Peripheral Nerve Damage
Peripheral axonal injury changes the expression of hundreds of genes in dorsal root ganglion neurons, increasing the excitability of the injured neurons and altering their synaptic transmission and connectivity in the dorsal horn of the spinal cord. We hypothesize that the regulation of genes by injury is orchestrated by induction of transcription factors that act as "master switches" to control coordinated changes in expression of programs of diverse genes. Specifically, we propose that activating transcription facter 3 (ATF3) is one of the master regulators of the altered phenotype induced by axonal injury. To study the nature and regulation of the transcriptional component of the responses neurons to injury and the particular involvement of ATF3 we propose to: 1. Test if ATF3 expression in DRG neurons is necessary after peripheral nerve injury and sufficient in non-injured states, to alter sensory behavior. To do this we will investigate the effect on pain sensitivity in the spared nerve injury neuropathic pain model of both knockdown ATF3 expression, by AAV8 delivery of shRNA to DRG neurons, and of conditional ATF3 knockout limited to adult DRG neurons, using an inducible Brn3a Cre driver. We will also examine if forced expression of ATF3 in postnatal non-injured DRG neurons increases pain-related sensitivity, using transgenic mice with the AFT3 gene under control of a neuron-specific thy1.2 regulatory sequence. 2. Establish if ATF3 contributes to the pain phenotype after nerve injury by altering the expression of functional-and growth-related genes. This will involve comparing microarray expression profiles of DRGs from intact and nerve-injured ATF3 overexpressing and knockout mice with wild types, and identification of those genes to which ATF3 finds in injured DRGs using chromatin immunoprecipitation. Validation of regulation by ATF3 will be by luciferase reporter assay and the role of candidate genes in neuropathic pain evaluated. 3. Determine if central sprouting of A-fiber afferents into the superficial laminae of the dorsal horn occurs after peripheral nerve injury and is driven by AFT3 expression. To do this we will use a transgenic mouse engineered to express an axonally transported GFP fusion protein only in large myelinated DRG neurons (using a Parv-Cre driver) in order to label the central terminals of these A-fibers. We find in pilot studies that peripheral nerve injury results in the sprouting of A-fiber central terminals into lamina II outer and will now both extend this and test if ATF3 contributes, using knockdown and knockout strategies. We will compare the pattern of structural plasticity in adult and immature nervous systems.