Figure 3. Shaping and mapping functional relay circuits.

Illustrated is a proposed approach to guide the formation of adaptive sprouting and map the resultant relay circuit by combining post-injury rehabilitation, retrograde trans-synaptic labeling, tissue clearing, and three-dimensional imaging of cleared brain and spinal cord. (A) In an uninjured animal, retrograde trans-synaptic tracing with fluorescent label from spinal motor neurons (green) –as can be achieved by injection of viral tracer into the muscle of interest (brown)– followed by tissue clearing to reveal proper connection of spinal motor neurons to corticospinal neurons (blue) in the corresponding motor cortex. Rodent spinal interneurons are not shown. (B) In a spinally injured animal (red “X”), spontaneous sprouting occurs but is undirected, leading to nonfunctional or maladaptive connections (indicated by blue arrow) that may change cortical motor representation and worsen motor function among other adverse effects. (C) In a spinally injured animal subjected to post-injury rehabilitation (exercise and/or electrochemical stimulation), spontaneous sprouting occurs and training strengthens adaptive circuits (indicated by blue arrow) exemplified by the establishment of relay connections to motor neurons through spinal interneurons (purple) to enhance functional recovery. Retrograde transneuronal tracing and tissue clearing allow subsequent visualization of such functional relay network. Furthermore, molecular interventions may enhance the sprouting response that in combination with rehabilitation could increase formation of new functional connections (not shown).