Methodology for quantifying excitability of identified projection neurons in the dorsal horn of the spinal cord, specifically to study spinal cord stimulation paradigms.

Using in and ex vivo preparations, electrophysiological methods help understand the excitability of biological tissue, particularly neurons, by providing microsecond temporal resolution. However, for in vivo recordings, in the context of extracellular recordings, it is often unclear precisely which type of neuron the tip of the electrode is recording from. This is particularly true in the densely-populated central nervous system, such as the spinal cord dorsal horn at both superficial and deep levels. Here, we present a detailed protocol for the identification of superficial dorsal horn spinal cord neurons that receive peripheral input and project to the brain, using multiple surgical laminectomies and the careful placement of electrodes. Once a superficial projection unit was found, quantification to electrical peripheral stimulation was performed using a Matlab algorithm to form a template of projection neuron response to controlled C2 stimulation and accurately match this to the responses from peripheral stimulation. These superficial spinal projection neurons are normally activated by noxious peripheral stimuli, so we adopted a well-characterised wind-up protocol to obtain a neuronal excitability profile. Once achieved, this protocol allows for testing specific interventions, either pharmacological or neuromodulatory (e.g., spinal cord stimulation) to see how these affect the neuron's excitability. This preparation is robust and allows the accurate tracking of a projection neuron for over 3-h. Currently, most existing methods record from dorsal horn neurons that are often profiled based on their excitability to different peripherally-applied sensory modalities. While this is well-established, it fails to discriminate between interneurons and projection neurons, which is important as these two populations signal via distinctly different neuronal networks. Using the approach detailed here will result in studies with improved mechanistic understanding of the signal integration and processing that occurs in the superficial dorsal horn. The refinements detailed in this protocol allow for more comprehensive studies to be carried out that will help understand spinal plasticity, in addition to many considerations for isolating the relevant neuronal population when performing in vivo electrophysiology.