Optogenetic Interrogation of Functional Synapse Formation by Corticospinal Tract Axons in the Injured Spinal Cord

To restore function after injury to the CNS, axons must be stimulated to extend into denervated territory and, critically, must form functional synapses with appropriate targets. We showed previously that forced overexpression of the transcription factor Sox11 increases axon growth by corticospinal tract (CST) neurons after spinal injury. However, behavioral outcomes were not improved, raising the question of whether the newly sprouted axons are able to form functional synapses. Here we developed an optogenetic strategy, paired with single-unit extracellular recordings, to assess the ability of Sox11-stimulated CST axons to functionally integrate in the circuitry of the cervical spinal cord. Initial time course experiments established the expression and function of virally expressed Channelrhodopsin (ChR2) in CST cell bodies and in axon terminals in cervical spinal cord. Pyramidotomies were performed in adult mice to deprive the left side of the spinal cord of CST input, and the right CST was treated with adeno-associated virus (AAV)-Sox11 or AAV-EBFP control, along with AAV-ChR2. As expected, Sox11 treatment caused robust midline crossing of CST axons into previously denervated left spinal cord. Clear postsynaptic responses resulted from optogenetic activation of CST terminals, demonstrating the ability of Sox11-stimulated axons to form functional synapses. Mapping of the distribution of CST-evoked spinal activity revealed overall similarity between intact and newly innervated spinal tissue. These data demonstrate the formation of functional synapses by Sox11-stimulated CST axons without significant behavioral benefit, suggesting that new synapses may be mistargeted or otherwise impaired in the ability to coordinate functional output.

As continued progress is made in promoting the regeneration of CNS axons, questions of synaptic integration are increasingly prominent. Demonstrating direct synaptic integration by regenerated axons and distinguishing its function from indirect relay circuits and target field plasticity have presented technical challenges. Here we force the overexpression of Sox11 to stimulate the growth of corticospinal tract axons in the cervical spinal cord and then use specific optogenetic activation to assess their ability to directly drive postsynaptic activity in spinal cord neurons. By confirming successful synaptic integration, these data illustrate a novel optogenetic-based strategy to monitor and optimize functional reconnection by newly sprouted axons in the injured CNS.