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Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, activating transcription factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65–80% by 16 d) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knock-out-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemizygotes and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed phosphatase and tensin homolog (PTEN) line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons (including putative spinothalamic and spinocerebellar) and descending supraspinal neurons (rubrospinal, vestibulospinal, reticulospinal and hypothalamic). We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes (e.g., via reporter expression), and in probing the function of any number of genes as they relate to neuronal injury and regeneration.

Dorsal root injury (DRI) disrupts the flow of sensory information to the spinal cord. Although primary afferents do not regenerate to their original targets, spontaneous recovery can, by unknown mechanisms, occur after DRI. Here, we show that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor or neurotrophin-4, are upregulated in the spinal gray matter after DRI. Because endognous BDNF and NT-3 have well established roles in synaptic and axonal plasticity, we hypothesized that they contributed to spontaneous recovery after DRI. We first developed a model of DRI-induced mechanosensory dysfunction: rat C7/8 DRI produced a deficit in low-threshold cutaneous mechanosensation that spontaneously improved within 10 d but did not recover completely. To determine the effects of endogenous BDNF and NT-3, we administered TrkB-Fc or TrkC-Fc fusion proteins throughout the recovery period. To our surprise, TrkB-Fc stimulated complete recovery of mechanosensation by 6 d after DRI. It also stimulated mechanosensory axon sprouting but prevented deafferentation-induced serotonergic sprouting. TrkC-Fc had no effect on low-threshold mechanosensory behavior or axonal plasticity. There was no mechanosensory improvement with single-bolus TrkB-Fc infusions at 10 d after DRI (despite significantly reducing rhizotomy-induced cold pain), indicating that neuromodulatory effects of BDNF did not underlie mechanosensory recovery. Continuous infusion of the pan-neurotrophin antagonist K252a also stimulated behavioral and anatomical plasticity, indicating that these effects of TrkB-Fc treatment occurred independent of signaling by other neurotrophins. These results illustrate a novel, plasticity-suppressing effect of endogenous TrkB ligands on mechanosensation and mechanosensory primary afferent axons after spinal deafferentation.