Long-term optical imaging of the spinal cord in awake behaving mice

Advances in optical imaging and fluorescent biosensors enable study of the spatiotemporal and long-term neural dynamics in the brain of awake animals. However, methodological difficulties and fibrosis limit similar advances in the spinal cord. Here, to overcome these obstacles, we combined in vivo application of fluoropolymer membranes that inhibit fibrosis, a redesigned implantable spinal imaging chamber and improved motion correction methods that together permit imaging of the spinal cord in awake behaving mice, for months to over a year. We demonstrated a robust ability to monitor axons, identified a spinal cord somatotopic map, performed months-long imaging in freely moving mice, conducted Ca²⁺ imaging of neural dynamics in behaving mice responding to pain-provoking stimuli and observed persistent microglial changes after nerve injury. The ability to couple in vivo imaging and behavior at the spinal cord level will drive insights not previously possible at a key location for somatosensory transmission to the brain.